UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · Gian, Claudinha, Josie, Josi e todos pelos quais...
Transcript of UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · Gian, Claudinha, Josie, Josi e todos pelos quais...
MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
SÍNTESE E AVALIAÇÃO DA ATIVIDADE ANTITUMORAL DE NANOGÉIS DE
FUCANA A DA ALGA MARROM Spatoglossum schröederi (C.Agardh)
Kützing
JAILMA ALMEIDA DE LIMA
NATAL/RN 2014
JAILMA ALMEIDA DE LIMA
SÍNTESE E AVALIAÇÃO DA ATIVIDADE ANTITUMORAL DE NANOGÉIS DE
FUCANA A DA ALGA MARROM Spatoglossum schröederi (C.Agardh)
Kützing
Tese apresentada ao Programa de Pós-
Graduação em Ciências da Saúde da
Universidade Federal do Rio Grande do
Norte como requisito para a obtenção do
título de Doutor em Ciências da Saúde.
Orientador: Prof. Dr. Hugo Alexandre de O. Rocha
NATAL/RN 2014
CATALOGAÇÃO NA FONTE
L732s
Lima, Jailma Almeida de.
Síntese e avaliação da atividade antitumoral de nanogéis de
fucana A da alga marrom Spatoglossum schöederi (C. Agardh)
Kützing / Jailma Almeida de Lima. – Natal, 2014.
106f. : il.
Orientador: Prof. Dr. Hugo Alexandre de O. Rocha.
Tese (Doutorado) – Programa de Pós-Graduação em Ciências
da Saúde. Centro de Ciências da Saúde. Universidade Federal do
Rio Grande do Norte.
1. Fucanas – Tese. 2. Polissacarídeos sulfatados – Tese.
3. Atividade antitumoral – Tese. 4. Nanogéis – Tese. I. Rocha,
Hugo Alexandre de O. II. Título.
RN-UF/BS-CCS CDU: 582.272(043.2)
ii
MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenador do Programa de Pós-Graduação em Ciências da Saúde:
Prof. Dr. Eryvaldo Sócrates Tabosa do Egito
iii
JAILMA ALMEIDA DE LIMA
SÍNTESE E AVALIAÇÃO DA ATIVIDADE ANTITUMORAL DE NANOGÉIS DE
FUCANA A DA ALGA MARROM Spatoglossum schröederi (C.Agardh)
Kützing
Aprovada em: 21 / 03 / 2014
Banca Examinadora:
Presidente da Banca:
Prof. Dr. Hugo Alexandre de Oliveira Rocha (UFRN)
Membros da Banca
Profa. Dra Valéria Soraya de Farias Sales (UFRN)
Prof. Dr. Artur da Silva Carriço (UFRN)
Profa. Dra. Valquíria Pereira de Medeiros (UFJF)
Profa. Dra. Norma Maria Barros Benevides (UFC)
iv
Dedico esta obra
A Deus.
Só tenho que agradecer-Te. Obrigada, Senhor, por tudo!
Grande é a sua Bondade e Misericórdia!
A minha Mãe, Francisca (Rosa).
Agradeço-te por tudo, especialmente pelo Amor que sempre me deste de forma
incondicional. EU TE AMO!
v
Dedico esta obra
A Hugo Rocha,
O meu eterno agradecimento por ser um educador nato, e por proporcionar não só a
mim, mas todos ao seu redor, a possibilidade de crescimento e busca por algo melhor.
Obrigada, Hugo, por tudo!
A Família BIOPOL,
Obrigada a todos àqueles que fazem ou que já fizeram parte desta história...
Dayanne (Dayn ou amigan), Mariana, Sara, Karol, Cinthia, Leandro, Diego (Popó),
Ruth, Rafael, Gabriel, Moacir, Raniere, Joanna, Letícia, Kaline, Arthur, Vinicius, Max,
Rony, Marília, Monique, Ajax, Fred, Larisse, Regina, Sarah (pequena), Mônica, Pablo,
Danielle, Almino Afonso, Jéssica, Mariane, Fernanda (Pôia), Leonardo Nobre (Leo),
Profa. Fabiana Lima, Ana Karina, Ana Karinne (Donana), Daniel, Fernando, Ivan,
Nednaldo, Eduardo, Edjane, Valquíria.
vi
Dedico esta obra
A “amigannn” Dayn,
Obrigada pela amizade, pelo companheirismo e por ter me proporcionado fazer parte
de sua família. Obrigada também pela confiança, bem mais que isso, pelo privilégio de
ser madrinha de seu filho Heitor e suplente de Helena!
A NOSSA AMIZADE É
Mais que uma mão estendida,
mais que um belo sorriso,
mais do que a alegria de dividir,
mais do que sonhar os mesmos sonhos
ou doer as mesmas dores,
muito mais do que o silêncio que fala
ou da voz que cala para ouvir
é a amizade, o alimento
que nos sacia a alma
e nos é ofertado por alguém
que crê em nós!
vii
Agradecimentos especiais
À UFRN, à Pós-graduação em Ciências da Saúde e ao Departamento de Bioquímica pela oportunidade de concluir esse curso de Pós-graduação, assim como as agências
Financiadoras CAPES e CNPq.
Agradeço novamente ao meu orientador Prof. Dr. Hugo Rocha pela oportunidade oferecida, pela atenção e auxílio prestados durante a pesquisa.
A todos os professores do CCS (UFRN), aos coordenadores do programa de pós-
graduação (PPGCSa) e às secretárias do programa.
As professoras da banca de qualificação: Profa. Naisandra Bezerra e Profa. Ivonete Araújo
A todos os professores do DBQ (UFRN), em especial, a Profa. Edda Lisboa Leite, pela sua força e por toda a sua contribuição à instituição. Obrigada por sempre ter
participado e me proporcionado grandes ensinamentos, principalmente de vida!
Aos meus amigos de laboratório pela colaboração e ajuda nos meus experimentos e também em tudo que precisei:
A Dayn (“amigann” e comadre) que tanto amo por me compreender e por sempre ter uma palavra amiga. Tenho por ti um enorme carinho. Obrigada por fazer parte de sua vida e de
seus flhos Helena e Heitor e daqui a alguns meses de Heloísa. A Karol, por ser esse doce de pessoa, alguém muito especial para mim! Tenho certeza
que seu futuro será brilhante e, mais que isso, queria agradecer-te por sempre estar perto nas horas mais difíceis com carinho e compreensão! Você sempre deixa o laboratório mais
alegre.
A Cinthia. Você é meu exemplo de transformação! Obrigada por tudo, obrigada por ter confiado a mim a tarefa tão importante de ser madrinha (casamento) e mais que isso, de
poder fazer parte da sua vida e de conviver com seu filho Davi. Obrigada A Ruth (Lut Lut), a “safada” que amo de paixão, a Rafael (super Rafildo), que não é o
ABC, mas é o mais querido, Leandro (Lelê) exemplo de profissional e de amigo.
Agradeço também de forma especial a Mariana e a Sara, por terem sido as primeiras a me incentivarem a vir para a Bioquímica. A Mariana, por me apoiar, por estar comigo em tudo que preciso, por me fazer ver meus erros, por ser essa grande amiga, te adoro! A
Sara, por ser nosso pilar de conhecimento “nosso Google”, aquela que nos socorre sempre e em qualquer tempo, te adoro muito. Muito obrigada, amigas, vocês são demais e
muito importantes para mim, estarão sempre no meu coração!!!
Agradeço muito a todos, todos vocês são mais que especiais nessa trajetória, a vocês o meu eterno agradecimento: Gabriel, Moacir, Joanna, Pablo, Raniere, Fernanda (Pôia), Leonardo Nobre (Leo), Letícia, Kaline, Arthur, Vinicius, Max, Rony, Marília, Monique,
Ajax, Fred, Larisse, Regina, Sarah (Pequena), Mônica, Danielle, Almino Afonso, Jéssica, Profa. Fabiana Lima, Ana Karina, Ana Karinne (Donana), Daniel, Fernando,
Ivan, Diego (Popó) e Valquíria.
Agradeço de forma especial a todos os amigos que fiz aqui no Departamento de
Bioquímica: Adriana Brito, Ana Katarina, Luciana Rabêlo, Jonalson, Anderson (Negão), Paula Ivani (Paulinha), Antônio, Marina, Ingrid, Lívia, Ana Katarina, Jefferson, Rômulo, Ana
Luiza, Paula e Demetrius, Juliana, Roberta, Thuany,
viii
Aos amigos dos laboratórios LAMA e LBMG. Conheci pessoas maravilhosas e que me ajudaram bastante no desenvolvimento de algumas técnicas: Beatriz Mesquita, Susana,
Nilmara, Paula Anastácia, Rita, Mayara, Jana, Felipe, Isabel, Dani (Pôia branca), Leonam
Agradeço a mais nova professora da genética, Susana Moreira, você superou todas as
dificuldades e chegou lá, que bom que ficarás por aqui. Tu és giro!!!
A todos que me ajudaram direta ou indiretamente nesta tese: Danilo Cavalcanti, Karla (Farmácia), Priscyla (UFPE), Guiman, Marina e Haroldo.
A Lurdinha, pessoa batalhadora, de uma humildade e sabedoria enorme. Você é uma pessoa
especial e que ensinou muito, não só a parte laboratorial, mas principalmente sobre a vida. Sou
muito grata pelos seus ensinamentos e pelos momentos “felizes” que compartilhamos. Muito
obrigada por tudo.
Agradeço, de forma especial, àqueles que contribuíram de forma diferencial para minha formação como pessoa: aos meus grandes amigos e amigas Joanna D'arc e sua filha
Joyce (que acompanhamos seu crescimento), Sara, Mariana Santana, Chrístier e Railson, vocês foram uma das melhores conquistas, todos vocês são formidáveis e pessoas muito
especiais pra mim. Muito obrigada por me aturarem e me aceitarem como sou. Muito obrigada do fundo do coração. Agradeço também a Adaíres e a Wanessa por fazerem
parte dessa história.
Agradeço à família de Dayn: Leonardo Oliveira (Leo), Dona Célia, Seu José, França, Drielle, Dmitryev, Dastaev (Patrícia e Monick), Dmetryus, Seu Abelardo, Helena, meu
afihado Heitor, Heloísa e a Dona Ceiça. Muito obrigada pela força e pelo carinho. Também dedico esta tese a vocês!
Muito obrigada a todos que fazem parte da Ong “Vida é Alegria”: Mileide, dona Neide, Sara, Mariana, Adaíres, Ricardo, Narjara, Adriana Sabiana, Adineide, Jobson, Fernando, Gian, Claudinha, Josie, Josi e todos pelos quais tenha esquecido o nome, mas que fazem
parte desse grupo pela oportunidade de ajudar a tantas crianças!
Agradeço a todos os amigos que trabalham comigo no CRI Inês, Josinete, Patrícia, Elias, Mércia, Lúcia, Ana Tereza, Severina, Lidiane, Adriana Pinto,
Sueldo, Cimária e Tarciana por compreenderem e me apoiarem nessa batalha que foi o doutorado.
Agradeço a Tarciana, minha companheira do coração, muito obrigada pelo apoio e por ser tão especial para mim. Agradeço também a todos da sua família: Taísa, Vanessa, Rosa,
Maria, Seu Tarcísio e Antônio.
Agradeço também de forma especial aos grandes amigos Eutália e André. Vocês são dois anjos que Deus colocou no mundo, não tenho palavras para descrever
como sou feliz por ter vcoês como amigos!
Agradeço a minha “mãe” Vivi e a Mari também por, apesar de longe, estarem tão perto de mim.
Como foi difícil escrever essa parte! Por mais que eu possa agradecer, ainda assim seria
muito pouco, não há como agradecer pelas alegrias, pelo apoio, pelo carinho, pelo consolo
nas frustações. São muitas as pessoas a quem gostaria de agradecer, mas poderá ser que
ao longo dos agradecimentos a memória possa esquecer de uma ou outra pessoa, mas
que não as tornam menos importantes!
ix
“Talvez não tenha conseguido fazer o melhor, mas
lutei para que o melhor fosse feito. Não sou o que
deveria ser, mas Graças a Deus, não sou o que era
antes”.
(Marthin Luther King)
x
RESUMO
Fucanas são polissacarídeos sulfatados encontrados em algas marrons e equinodermos. Tem sido demonstrado que uma fucana denominada de fucana A, obtida da alga marrom Spatoglossum schröederi, apresenta uma série de efeitos biológicos, em particular, a atividade antitumoral. Com intuito de se potencializar essa atividade, foram adicionados grupamentos tióis a estrutura da fucana A. Posteriormente, os nanogéis foram sintetizados pela formação de nanocomplexos entre a fucana A tiolada e o polietileno glicol (PEG) em várias relações 2.5, 5.0, 10, 15 e 30. Os nanogéis com as relações de 10 e 15 (FucA:PEG10 e FucA:PEG15) foram os que se apresentaram com os menores tamanhos, mais esféricos, com diâmetro em torno de 186,95 ± 10,62 nm e carga de superfície ligeiramente negativa. Após a síntese dos nanogéis, estes foram submetidos aos ensaios antiproliferativos com células da linhagem tumoral 786-0 nas concentrações 8,0 a 64 µg/mL. As células foram analisadas durante um período de 24, 48 e 72 horas. Os dados mostraram que em todas as concentrações de nanogéis de fucana A, a atividade antiproliferativa foi tempo e dose dependente, o mesmo não sendo observado para a fucana A avaliada isoladamente. O nanogel de FucA:PEG15 também induziu apoptose por mecanismos dependentes e independentes de caspases. Posteriormente, FucA:PEG15 também foi marcado com FITC sendo completamente incorporado pelas células 786-0 após 1 hora. Quando a endocitose celular foi parada, o FucA:PEG15 teve o seu efeito antiproliferativo reduzido. Apesar de FucA:PEG15 não possuir efeito anticoagulante por aPTT e PT (até 100 µg/mL), ele apresesentou efeito antioxidante e angiogênico. Esses dados mostram que o nanogel de fucana A exibe várias efeitos (antiproliferativa, antioxidante e antiangiogênica) e, portanto, o seu potencial para a terapia do câncer deve ser investigada.
Palavras chaves: Polissacarídeos sulfatados, fucanas, nanogéis, atividade
antitumoral, citotoxicidade.
xi
LISTA DE ABREVIATURAS E SIGLAS
µL Microlitros
786-0 Linhagem de células derivadas de adenocarcinoma renal
aPTT Tempo de tromboplastina parcialmente ativada
B16-F10 Linhagem de células de melanoma murino
CAT Capacidade antioxidante total
CO2 Dióxido de carbono
DAPI 4′,6′-diamino-2-fenilindol
DAPI 4',6-diamidino-2-phenylindole
DLS Dynamic light scattering
DMEM Meio de cultura sintético complexo – Dubelcco’s Modified Eagle’s Medium
DMSO Dimetilsulfóxido
DPPH 2,2-difenil-1-picrilhidrazila
ECs Células endoteliais ativadas
EHS Tumor Engelbreth-Holm-Swarm
F0.5 Fração precipitada com 0,5 volumes de acetona
F0.6 Fração precipitada com 0,6 volumes de acetona
F0.7 Fração precipitada com 0,7 volumes de acetona
F0.9 Fração precipitada com 0,9 volumes de acetona
F1.1 Fração precipitada com 1,1 volumes de acetona
F1.3 Fração precipitada com 1,3 volumes de acetona
F2.0 Fração precipitada com 2,0 volumes de acetona
FITC Isotiocianato de fluoresceína
Fuc A Fucana A
g Grama
G0 Fase do ciclo celular em que a célula permanece indefinidamente na intérfase
HeLa Linhagem de células de carcinoma cervical humano
HepG2 Linhagem de Células de hepatocarcinoma humano
HS-5 Linhagem de células estromais da medula óssea humana
kDa Kilodalton
M Molar
MEV Microscópio eletrônico de varredura
mg Miligrama
Mili-Q Água ultrapura
Min. Minutos
mL Mililitros
mM Milimolar
mm Milímetros
MTT 3-(4,5-dimethylthiazol-2-y1)2,5-diphenil tetrazolium bromide)
xii
MW Peso molecular
nm Nanômetros
PA Para análise
Panc-1 Linhagem de células de adenocarcinoma de pâncreas
PBS Solução tampão de salino fosfato
PDA Tampão 1,3 diamino propano acetato
PEG Polietileno glicol
pH Potencial de hidrogênio
PI Iodeto de propídio
PT Tempo de protrombina
RAEC Linhagem de células endoteliais de aorta de coelho
RPMI Meio de cultura sintético complexo criado pelo Roswell Park Memorial Institute
SFB Soro fetal bovino
xiii
LISTA DE FIGURAS Figura 1. Alga marrom S. schröederi (C. Agardh) Kützing. A) em exsicata (Foto:
Nednaldo Dantas) e B) na natureza (Foto: Edjane Barroso)..................... 21
xiv
SUMÁRIO
RESUMO................................................................................................................................... x LISTA DE ABREVIATURAS E SIGLAS..................................................................................... xi LISTA DE FIGURAS.................................................................................................................. xiii 1. INTRODUÇÃO....................................................................................................................... 15 2. JUSTIFICATIVA.................................................................................................................... 18 3. OBJETIVOS........................................................................................................................... 19
3.1. OBJETIVO GERAL................................................................................................. 19 3.2. OBJETIVOS ESPECÍFICOS.................................................................................. 19
4. MÉTODOS............................................................................................................................. 20 4.1. MATERIAIS BIOLÓGICOS..................................................................................... 21
4.1.1. Algas...................................................................................................... 21 4.1.2. Linhagens e culturas celulares........................................................... 22
4.2. EXTRAÇÃO E PURIFICAÇÃO DA FUCANA A...................................................... 22 4.2.1. Obtenção do pó cetônico..................................................................... 22 4.2.2. Proteólise.............................................................................................. 22 4.2.3. Fracionamento do extrato bruto com concentrações crescentes
de acetona............................................................................................. 23
4.2.4. Cromatografia em coluna de troca iônica.......................................... 23 4.3. SÍNTESE E CARACTERIZAÇÃO DOS NANOGÉIS.............................................. 24
4.3.1. Síntese das fucanas tiolada................................................................. 24 4.3.2. Caracterização físico-química dos nanogeis de fucanas................. 24 4.3.3. Transmitância dos nanogéis............................................................... 24 4.3.4. Dynamic Light Scattering (DLS).......................................................... 25 4.3.5. Estabilidade........................................................................................... 25 4.3.6. Microscopia eletrônica de varredura (MEV)....................................... 25 4.3.7. Microscopia confocal........................................................................... 25 4.3.8. Espectroscopia de infravermelho....................................................... 26 4.3.9. Atividade antiproliferativa.................................................................... 26 4.3.10. Conjugação da fucana A com fluoresceína (FITC).......................... 27 4.3.11. Avaliação da viabilidade e morte celular por anexina V-FITC/
iodeto de propídio (PI)....................................................................... 27
4.3.12. Atividade antioxidante....................................................................... 28 4.3.13. Atividade anticoagulante................................................................... 28 4.3.14. Ensaio de formação de tubo de matrigel......................................... 28
4.4. ANÁLISE ESTATÍSTICA........................................................................................ 29
5. ARTIGOS PRODUZIDOS...................................................................................................... 30 5.1. ARTIGO 1 (SUBMETIDO)...................................................................................... 32 5.2. CAPÍTULO DE LIVRO............................................................................................ 55 5.3. ARTIGO 2............................................................................................................... 77 5.4. ARTIGO 3............................................................................................................. 83
6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...................................................................... 91 7. REFERÊNCIAS..................................................................................................................... 93 8. ANEXOS................................................................................................................................ 96
8.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)............................................... 97 8.2. NORMAS DA REVISTA PARA SUBMISSÃO (MARINE DRUGS)......................... 100 8.3. DECLARAÇÃO....................................................................................................... 105 8.4. COMITÊ DE ÉTICA................................................................................................ 106
15
Almeida-Lima J. PPGCSA/CCS
1. INTRODUÇÃO
O câncer é um dos problemas mais complexos que os sistemas de saúde
mundial enfrentam e essa doença está prestes a se tornar uma das maiores
causas de mortalidade nas próximas décadas. Segundo a Organização
Mundial de Saúde (WHO) o número de casos de câncer no mundo deverá
aumentar em 75% até 2030. E segundo esta mesma pesquisa, essa taxa pode
ser ainda mais alta e chegar a 90% em países mais pobres [1, 2].
Para o tratamento do câncer, os três principais tratamentos atuais são a
cirurgia, a radioterapia e a quimioterapia, cuja escolha depende do tipo de
tumor e do estágio de seu desenvolvimento [3]. Embora esses tratamentos
sejam de grande valor, podem apresentar desvantagens e limitações, como
complicações pós-cirúrgicas e toxicidade sistêmica. Por essa razão, pesquisas
que buscam métodos alternativos e/ou complementares de tratamento estão
em evidência e visam sempre ser mais eficientes em relação às terapias
convencionais [4].
Atualmente, a nanotecnologia exibe um indispensável papel
especialmente no campo da medicina (nanomedicina) e vem sendo apontada
como uma das grandes promessas do futuro. As abordagens nanoterapêuticas
têm tratado diferentes tipos de câncer e têm tornado possível uma nova era na
quimioterapia, já que vários tipos de nanoestruturas (dendrímeros,
nanossondas magnéticas, nanoesferas, nanopartículas, hidrogéis, lipossomos,
dentre outros) têm sido sintetizados para atingir as células cancerosas, tanto
para o diagnóstico quanto para terapias específicas [5, 6], já que esses
sistemas nanométricos oferecem a vantagem de reduzir ou eliminar efeitos
colaterais da quimioterapia por atuarem diretamente nas células cancerosas e
não permanecerem livres na via sistêmica.
Dentre os nanossistemas sintetizados, os nanogéis atualmente emergem
como um grupo capaz de interagir especificamente com células cancerígenas.
Nanogéis usualmente são definidos como dispersões aquosas de
nanopartículas formadas por polímeros química ou fisicamente interligados.
16
Almeida-Lima J. PPGCSA/CCS
Além disso, eles têm atraído crescente interesse devido o seu potencial como
nanocarreadores de vários compostos, dentre eles os biopolímeros [7, 8].
Entre a numerosa quantidade de biopolímeros que tem sido proposta para
a preparação de nanogéis, polissacarídeos têm inúmeras vantagens sobre os
polímeros sintéticos por serem não tóxicos, biocompatíveis, biodegradáveis e
solúveis em água [9]. Ao longo dos últimos anos, um polímero em especial tem
chamado a atenção na área dos polissacarídeos, é conhecido como fucana.
Fucana é um termo utilizado para denominar uma família de polissacarídeos
sulfatados cujo açúcar mais representativo é a α-L-fucose sulfatada. Elas são
encontradas em algas marrons e em equinodermas (ouriço e pepino do mar)
[10, 11].
Ao longo de algumas décadas nosso grupo de pesquisa (localizado no
Laboratório de Biotecnologia de Polímeros Naturais – BIOPOL – UFRN, sob a
responsabilidade do Prof. Dr. Hugo Rocha) tem intensificado os estudos com
os polissacarídeos extraídos da alga marrom Spatoglossum schröederi
(Dictyotaceae). Essa alga sintetiza três tipos de fucanas e a obtida em maior
quantidade foi nomeada de fucana A [12, 13]. A disponibilidade permanente
desse organismo em grandes quantidades tornou-a uma excelente escolha
para a prospecção de compostos bioativos.
Em estudos anteriores, Barroso e colaboradores trabalhando com a
fucana A da S. schröederi observaram que esse polímero não apresentava
atividade anticoagulante in vitro, porém, demonstrou atividade antitrombótica in
vivo, sendo observado um efeito dose-dependente alcançando a saturação ao
redor de 20 µg/g de peso de rato. A fucana A também apresentou um efeito
tempo-dependente, alcançando a saturação por volta de 16h após a sua
administração [13].
Como essa atividade antitrombótica apresentada pela fucana A foi de
enorme importância farmacológica, testes com esse polímero continuaram a
ser realizados na tentativa de preencher as lacunas que ainda faltam para,
quem sabe, em um futuro próximo, a fucana possa ser utilizada como um
fármaco. Estudos para verificar a toxicidade da fucana A foram investigados.
Testes de toxicidade in vivo com ratos Wistar [14] e in vitro para observar a
17
Almeida-Lima J. PPGCSA/CCS
genotoxicidade [15] foram realizados e não mostraram nenhum efeito danoso
provocado pela fucana A, mesmo utilizando altas concentrações. Ainda neste
mesmo trabalho, a atividade citotóxica da fucana A foi testada contra várias
linhagens tumorais, onde foi observado que esse polímero inibiu a proliferação
celular em torno de 43,7% para as células Panc-1 e HeLa (0,05 a 1 mg/mL) e
que ele não matou células normais.
A citotoxicidade para células tumorais também foi encontrada para a
heparina (polissacarídeo sulfatado de origem animal com estrutura química
semelhante a da fucana). Esse polímero demonstrou atividade antiproliferativa
frente a uma gama de linhagens celulares [16, 17].
Esse grande interesse pelas fucanas de algas pode estar relacionado
com a sua semelhança estrutural com a heparina, o que daria a esse
polissacarídeo atividades semelhantes às deste glicosaminoglicano. Além
disso, por serem de origem vegetal, elas poderiam apresentar menores riscos
de contaminações e são encontradas em abundância na natureza, já que são
recursos naturais renováveis.
No caso da heparina, têm sido desenvolvidos sistemas de nanogéis que
são resistentes ao ambiente extracelular e que não se degradam no interior
celular, promovendo a liberação controlada da heparina no interior das células
tumorais e, por conseguinte, a morte celular por apoptose induzida pela
heparina. Assim, quando Bae e colaboradores, utilizando nanogéis de
heparina, trataram células tumorais B16-F10, observaram que a proliferação
celular foi inibida em cerca de 50%, enquanto que a heparina sozinha inibiu o
crescimento celular em aproximadamente 10% [18].
Inicialmente nanogéis de fucana A da S. schröederi foram produzidos pela
conjugação da fucana a hexadecilamida. O nanogel de fucana A produzido
mostrou tamanho médio de 123 nm, carga negativa e estabilidade química (70
dias). Em seguida, o teste de citotoxicidade desse nanogel foi realizado contra
as linhagens tumorais HepG2, 786-0, HS-5, sendo a 786-0 a que apresentou
maior inibição, com aproximadamente 43,7% (0,05 a 0,5 mg/mL). Entretanto,
esse tipo de nanogel tem algumas limitações, pois tem um ambiente interno
lipofílico, como também há uma modificação estrutural considerável da fucana,
18
Almeida-Lima J. PPGCSA/CCS
o que pode alterar suas atividades [19]. Portanto, outras técnicas de síntese de
nanogéis de fucanas devem ser testadas a fim de se obter nanogéis de
fucanas que apresentem as atividades da fucana A.
19
Almeida-Lima J. PPGCSA/CCS
2. JUSTIFICATIVA
O Estado do Rio Grande do Norte possui uma grande diversidade de
espécies de macroalgas marinhas, organismos estes que são potências
produtores de compostos com grande potencial farmacológico e biotecnológico.
Dentre eles, destacam-se os polissacarídeos sulfatados. Apesar do potencial
dos polissacarídeos sulfatados encontrados em nossa região, esses compostos
ainda não são conhecidos, o que faz com que esses recursos naturais não
sejam aproveitados.
Recentemente, foram encontradas fucanas com alta atividade
antitumoral, sintetizadas por algas do litoral potiguar e o nosso grupo de
pesquisa vem se dedicando a pesquisar as fucanas dessas algas. Mas, apesar
da forte atividade antiproliferativa já encontrada em algumas fucanas de algas
marrons, há um empecilho que dificulta os avanços dos estudos com fucanas
antiproliferativas que é o seu caráter iônico, elas podem assim se ligar a uma
gama de proteínas extracelulares antes de entrarem no interior celular, o que
exige uma elevada concentração de fucanas para que elas possam
desempenhar o seu efeito.
Devido a isso, a síntese de nanogéis de fucana foi o recurso utilizado
neste trabalho para intensificar os estudos de suas atividades biológicas,
especialmente, o seu efeito antitumoral, já que os nanogeís são liberados
(introduzidos) diretamente dentro das células tumorais, sem serem “perdidos
na circulação”. Para tal, uma parceria foi estabelecida com a Universidade do
Minho (Portugal), centro de referência na produção de nanogéis, o que
viabilizou o desenvolvimento desses nanogéis aqui no Brasil.
20
Almeida-Lima J. PPGCSA/CCS
3. OBJETIVOS
3.1. GERAL
Sintetizar um nanogel de fucana A pela adição de grupos tióis e avaliar seu
efeito antiproliferativo e apoptótico frente a linhagem tumoral 786-0, como
também seu efeito antioxidante, anticoagulante e angiogênico.
3.2. ESPECÍFICOS
Extrair os polissacarídeos sulfatados da alga marrom S. schröederi por
fracionamento cetônico;
Obtenção da fucana A por cromatografia de troca iônica da fração
cetônica F0.6v;
Sintetizar nanogéis a partir da fucana A obtida;
Caracterizar físico-quimicamente os nanogéis de fucana A;
Avaliar as atividades antiproliferativa, anticoagulante, antioxidante e
antiangiogênica dos nanogéis produzidos e da fucana A livre;
Avaliar a capacidade dos nanogéis de fucana em induzir apoptose em
células tumorais;
Verificar a sua internalização celular pela conjugação do FITC com os
nanogéis de fucana A.
21
Almeida-Lima J. PPGCSA/CCS
4. MÉTODOS
4.1. MATERIAIS BIOLÓGICOS
4.1.1. Algas
A alga marinha marrom Spatoglossum schröederi (C. Agardh) Kützing
(Figura 1) foi coletada na Praia de Búzios, município de Nísia Floresta (litoral
sul do Rio Grande do Norte), em marés baixas entre 0,0 a 0,2 metros a uma
temperatura situada entre 28-30°C. As algas foram recolhidas quando já
desprendidas do substrato, mas permanecendo flutuando nas águas de maré-
baixa.
As algas foram trazidas ao laboratório no mesmo dia da coleta e
acondicionadas em sacos de polietileno, lavadas em água corrente,
examinadas cuidadosamente para remoção de epífitas, inclusões calcárias e
sais, sendo postas para secar em estufa aerada a 45°C. Em seguida foram
trituradas, pesadas e guardadas em frascos de vidro hermeticamente fechados.
Figura 1 – Alga marrom S. schröederi (C. Agardh) Kützing. A) em exsicata (Foto:
Nednaldo Dantas) e B) na natureza (Foto: Edjane Barroso)
22
Almeida-Lima J. PPGCSA/CCS
4.1.2. Linhagens e culturas celulares
As linhagens celulares de adenocarcinoma renal (786-0) e de endotélio
da aorta de coelho (RAEC) foram mantidas em meio RPMI e HAM-F12,
respectivamente. Todas as células foram cultivadas a 37°C em uma incubadora
umidificada na presença de 5% CO2, com os meios suplementados com 10%
de soro fetal bovino (SFB) e antibióticos (100 U/mL de penicilina e 100 μg/mL
de estreptomicina). As células 786-0 foram doadas pela Profa. Dra. Carmen
Ferreira (Departamento de Bioquímica, UNICAMP, Brasil) e as RAEC pela
Profa. Dra. Helena Nader (Departamento de Bioquímica, UNIFESP, Brasil).
4.2. EXTRAÇÃO E PURIFICAÇÃO DA FUCANA A
4.2.1. Obtenção do pó cetônico
A alga seca e pulverizada foi suspensa em dois volumes de acetona PA
para despigmentação e delipidação do material. Essa solução ficou a
temperatura ambiente durante um período de 24 horas. Posteriormente, a
mistura foi decantada e o resíduo colocado para secar a 45°C sob aeração e
denominado de “pó cetônico”. Esse pó foi utilizado em seguida na proteólise.
4.2.2. Proteólise
Para a realização dessa etapa, foram adicionados dois volumes de NaCl
a 0,25 M ao pó cetônico (100 g) e o pH ajustado para 8,0 com NaOH. A esse
material foi adicionado a enzima proteolítica prozima (15 mg/g de pó cetônico).
Essa suspensão permaneceu em banho-maria a 60°C durante um período de
18h. Depois, foi filtrado e o sobrenadante submetido a uma centrifugação
10.000 x g por 15 minutos a temperatura de 4°C. Após a centrifugação, o
sobrenadante, que contém os polissacarídeos solúveis foi denominado de
extrato bruto de polissacarídeos sendo seco à pressão reduzida, triturado,
pesado e guardado para posteriores análises.
23
Almeida-Lima J. PPGCSA/CCS
4.2.3. Fracionamento do extrato bruto com concentrações crescentes de
acetona
O extrato polissacarídico bruto obtido foi fracionado com volumes
crescentes de acetona, obtendo-se as frações polissacarídicas. Os valores de
acetona adicionados foram determinados pela turvação da solução, que
caracteriza a precipitação de polissacarídeos devido à adição desse solvente
polar. Adicionou-se um volume de acetona, sob agitação leve, necessário para
que se visualizasse uma turvação da solução, essa solução foi mantida em
repouso a 4ºC durante 18h, o precipitado foi coletado por centrifugação a 8.000
x g por 15 minutos a 4ºC e seco a pressão reduzida.
Em seguida, esse procedimento foi repetido até que não se visualizasse
mais a formação de precipitado. As frações obtidas foram denominadas
conforme o volume de acetona no qual foram precipitadas (F0.5, F0.6, F0.7,
F0.9, F1.1, F1.3 e F2.0).
4.2.4. Cromatografia em coluna de troca iônica
A fração cetônica F0.6 (que contém a fucana A) foi submetida à
complexação com a resina de troca iônica Lewatite (10 mg de material para
cada 1,0 mL de resina) e a eluição foi realizada passo a passo utilizando-se
molaridades crescentes de NaCl, como descrito por Dietrich e colaboradores
[20]. Foram coletadas frações, com volume total de três vezes o volume da
resina, para cada molaridade de sal (0.3, 0.5, 0.7, 1.0, 1.5, 2.0, e 3.0 M), as
quais foram separadas pela ausência de positividade para o método de fenol-
ácido sulfúrico [21]. O fluxo de coleta foi de 1 mL/min, sendo o volume de
eluição igual para todas as molaridades coletadas.
As frações eluídas com 1.0 e 1.5 M de NaCl foram precipitadas com 2
volumes de metanol PA a 4°C e deixadas em repouso por 18h, sendo
posteriormente centrifugadas a 10.000 x g, por 15 minutos e secas a pressão
reduzida. Essas duas frações (1.0 e 1.5 M) são consideradas como detentoras
da fucana A [12, 13].
24
Almeida-Lima J. PPGCSA/CCS
4.3. SÍNTESE E CARACTERIZAÇÃO DOS NANOGÉIS 4.3.1. Síntese das fucanas tiolada As fucanas inicialmente foram dissolvidas em tampão citrato (0,1 M, pH
3.0) e postas para reagir com periodato de sódio por 2h a 4°C. Após esse
procedimento, as fucanas, agora tioladas, foram conjugadas com cisteamina
por aminação redutiva. Para tal, após diálise contra água destilada, as fucanas
modificadas reagiram com a cisteamina por duas horas em PBS. Após esse
período, foi acrescido à solução boridreto de sódio 0,1 M lentamente, sendo a
solução agitada por 1h a 4°C. Essa solução foi então dialisada sob atmosfera
de nitrogênio par minimizar a oxidação dos grupos tiois e posteriormente
liofilizada [18].
4.3.2. Caracterização físico-química dos nanogeis de fucanas As fucanas tioladas foram em seguida misturadas com polietileno glicol
(PEG) em diferentes proporções (2.5, 5.0, 10, 15 e 30), o que permitiu
posteriormente escolher qual a melhor proporção para a síntese do nanogel. A
mistura seca de fucana tiolada e PEG foi solubilizada em DMSO e incubada
por 6h a 37°C. A complexação entre a fucana e o PEG ocorreu através de
pontes de hidrogênio. Após esse período, a solução foi sonicada por 3 minutos
gerando pontes dissulfeto entre as moléculas de fucana tioladas. O nanogel
resultante foi exaustivamente dialisado e assim ficando livre das moléculas de
PEG e DMSO residuais.
4.3.3. Transmitância dos nanogéis
A transmitância da solução contendo nanogéis de fucana A foi
preparada usando várias razões em peso de PEG (0, 2,5, 5,0, 10, 15, 30). A
leitura foi medida a 400 nm com o leitor de microplacas Multiskan Ascent
(Thermo Labsystems Franklin, MA, EUA).
25
Almeida-Lima J. PPGCSA/CCS
4.3.4. Dynamic Light Scattering (DLS)
O diâmetro e o potencial zeta dos nanogéis foram medidos por
espectroscopia de correlação de fótons usando o equipamento DLS da
Brookhaven 90 Plus (Brookhaven Instruments Corporation, New York, USA).
Os nanogéis de fucana A foram preparados em água a 25°C a uma
concentração de 1 mg/mL.
4.3.5. Estabilidade
A estabilidade foi avaliada analisando o nanogel, solubilizado em água,
para a sua distribuição de tamanho. A análise foi realizada semanalmente, até
42 dias, conforme descrito anteriormente. A solução nanogel foi mantida a 4°C
durante o estudo e removidos para análise 24 horas antes de cada medição,
sempre realizada a 25°C.
4.3.6. Microscopia eletrônica de varredura (MEV)
Tamanho e forma dos nanogéis A fucana foram avaliados por
microscopia eletrônica de varredura. Para ambas as experiências, 50 µL da
solução de nanogel foi depositado sobre uma superfície limpa de mica, deixado
a secar a 25ºC e, em seguida, observada em microscópio de varredura
Shimadzu, modelo SSX550 (Shimadzu Scientific Equipment, UK).
4.3.7. Microscopia confocal
As células 786-0 (1 × 105) foram colocadas em lamínulas de vidro de 12
mm de diâmetro em placas de 24 wells (Nunc; Naperville, IL, USA). Após 3 dias
em cultura, as células foram lavadas três vezes com PBS (0,1 M, pH 7,4), e,
em seguida, tratadas com a fucana A ou com o nanogel de fucana marcado
com fluoresceína em meio RPMI isento de soro por 15, 30 ou 60 min a 37°C.
Em seguida, as células foram lavadas e fixadas com paraformaldeído a
2% (4°C). Após lavagem com PBS, os núcleos das células foram coradas com
solução de DAPI (50 µg/mL em PBS) durante 30 minutos, sendo lavadas cinco
26
Almeida-Lima J. PPGCSA/CCS
vezes em PBS novamente e em seguida adicionado Fluoromount-G (EM
Sciences; Ft. Washington, WA, EUA) e colocado uma lamínula de vidro e
examinadas com um microscópio confocal ou de fluorescência (Zeiss Axio
Examiner LSM 710, Jena, Alemanha).
4.3.8. Espectroscopia de infravermelho
A espectroscopia de infravermelho foi realizada em espectrômetro
Perkin-Elmer de 4400 a 400 cm-1 no Departamento de Química da
Universidade Federal do Rio grande do Norte. A fucana A e os nanogéis de
fucana A (~5 mg) foram analisados após secagem em aparelho de
Abdenhalden sob a forma de pastilha de KBr contendo P2O5 a 60ºC.
4.3.9. Atividade antiproliferativa
A atividade antiproliferativa dos nanogéis obtidos foi avaliada pelo ensaio
colorimétrico do MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenil tetrazolium
bromide) [22]. Esse método é baseado na redução do MTT a cristais de
formazan pelas células vivas.
Aproximadamente 1 x 104 células das linhagens 786-0 foram colocadas
em placa estéril de 96 wells para um volume final de 100 µL de meio RPMI
suplementado com 10% de soro fetal bovino. Após 24 horas, o meio foi
removido e as células foram carenciadas por 24 horas com meio sem soro.
Posteriormente, o meio foi aspirado e as células foram estimuladas a sair de
G0 pelo acréscimo de meio com SFB 10% na ausência (controle) e na
presença das amostras (8, 16, 32, 48 e 64 µg/mL). Após 24 horas de
tratamento, MTT (1 mg/mL) foi adicionado às células, e incubadas por mais 4
horas. Após esse período, o meio foi aspirado e adicionou-se 100 µL de etanol
PA para dissolver os cristais de formazan formados e precipitados. A
quantificação da absorbância foi feita em leitor de placa de 96 wells em
comprimento de onda de 570 nm. O ensaio foi realizado em triplicata. O cálculo
de inibição da proliferação celular foi realizado em comparação com o controle
contendo células não tratadas com as amostras.
27
Almeida-Lima J. PPGCSA/CCS
4.3.10. Conjugação da fucana A com fluoresceína (FITC)
A fim de visualizar a incorporação celular do nanogel (FucA:PEG15) e da
fucana A, ambas foram marcadas com sal de sódio de fluoresceína (Sigma).
Resumidamente, 5 mg de fucana A ou nanogel foram colocados para reagir
com 1 mg de fluoresceína em solução 0,1 M de PBS (pH 7,0), sendo agitada
durante 1 hora, protegida da luz, a temperatura ambiente. A solução foi
dialisada contra água destilada (MW 12 kDa) e, em seguida, liofilizada.
4.3.11. Avaliação da viabilidade e morte celular por anexina V-FITC/ iodeto
de propídio (PI)
Para avaliar a viabilidade e morte celular, foi utilizado um kit para
detecção de apoptose de Anexina V-FITC/PI, de acordo com as instruções do
fabricante, com pequenas modificações (BD Pharmingen, San Diego, CA). As
células 786-0 foram plaqueadas em uma concentração de 2 x 105 células em
placas de 6 wells. Após 24 horas para adesão das células, o meio foi removido
e as células foram carenciadas por 24 horas com meio sem soro.
Posteriormente, o meio foi aspirado e as células foram estimuladas a sair de
G0 pelo acréscimo de meio com SFB 10% na ausência (controle) e na
presença de nanogel (FucA:PEG15) e fucana A (64 µg/mL). Após o tratamento,
as células foram tripsinizadas, coletadas e lavadas duas vezes com PBS
gelado e ressuspendidas em 100 µL de tampão de ligação. Um total de 5 µL de
Anexina V-FITC e 5 µL de iodeto de propídio (PI) foram adicionados e a mistura
foi incubada por 30 minutos no escuro. Finalmente, 400 µL do tampão de
ligação foram adicionados às células, a suspensão celular foi analisada por
citometria de fluxo. A percentagem de células em apoptose foi determinada a
cada 10.000 eventos e os gráficos representam dados obtidos de três
experimentos separados. Para análise desses dados foi utilizado o programa
FlowJo® Analysis Software versão 9.3.2 (Tree Star Incorporation, OR, EUA).
28
Almeida-Lima J. PPGCSA/CCS
4.3.12. Atividade antioxidante
Quatro testes foram realizados para analisar a atividade antioxidante do
nanogel e da fucana A; foram eles: poder redutor, DPPH, sequestro de radicais
superóxido e capacidade antioxidante total. A metodologia foi seguida de
acordo com o descrito por Costa e colaboradores [23] e Vinayak, Sabu e
Chatterji [24].
4.3.13. Atividade anticoagulante
Os ensaios de tempo de tromboplastina parcial ativada (aPTT) e tempo
de protrombina (PT) foram realizados seguindo o protocolo fornecido pelos
“kits” comerciais adquiridos. Para esses ensaios foi utilizada uma massa de
100 µg para o nanogel (FucA:PEG15) e para a fucana A, sendo considerado o
possuidor de atividade aquela amostra capaz de prolongar em duas vezes o
tempo normal de coagulação. Foram utilizadas como meio de comparação da
atividade anticoagulante, a clexane (heparina de baixo peso molecular). Os
tempos de coagulação foram determinados utilizando-se um coagulômetro
automático.
4.3.14. Ensaio de formação de tubo de matrigel
O ensaio de formação do tubo foi adaptado a partir de Dreyfuss e
colaboradores [25]. A matrigel purificada a partir do tumor EHS foi
descongelada a 4°C e mantidas em gelo, cultivada em placas de 24 wells, e
incubadas a 37°C durante 16 h para gelificação. A ECs (105 células) foi
plaqueada no Matrigel em meio Ham-F12 contendo 10% de SFB em soro
fisiológico (controle) e em diferentes concentrações (12.5, 25, 50, e 100 µg/mL)
de nanogel (FucA:PEG15). As culturas foram mantidas a 3 °C em atmosfera
úmida de CO2 a 2,5% durante 24 horas. Cada tratamento foi realizado em
triplicata. A formação do tubo foi examinada sob um microscópio de luz
invertida em 50 X (ampliação). Três imagens foram tomadas de forma aleatória
em diferentes áreas.
29
Almeida-Lima J. PPGCSA/CCS
4.4. ANÁLISE ESTATÍSTICA
Todos os dados dos experimentos realizados foram expressos como
média ± desvio padrão. Foi utilizado o teste de análise paramétrica de análise
de variância (ANOVA) seguido do teste de Tukey (Nível de significância de
p<0,05) como GraphPad InStat® Software versão 3.05 para Windows 95
(GraphPad Software Incorporation, San Diego, CA, EUA).
30
Almeida-Lima J. PPGCSA/CCS
5. ARTIGOS PRODUZIDOS
5.1. Artigo 1 (SUBMETIDO)
Evaluation of potential antitumor activity of fucan nanogel
Periódico: Marine Drugs
Nanotechnology (The reference number for the article is NANO-102955)
Fator de impacto: 3.978
ISSN: 1660-3397 (Printed version)
ISSN: 1660-3397 (Online version)
Qualis: Medicina II – A2
Indexada: PubMed – indexado por MEDLINE
5.2. Capítulo de livro
Chapter 6 – Application of Marine Polysaccharides in Nanotechnology
Periódico: Marine Medicinal Glycomics
Biotechnology in Agriculture, Industry and Medicine Biochemistry
Research Trends
In: Vitor Hugo Pomin. (Org.). Marine Medicinal Glycomics. 1ed.New York: Nova
Science, 2013, v. 01, p. 65-114.
Binding: ebook
ISBN: 978-1-62618-649-1
5.3. Artigo 2
Evaluation of acute and subchronic toxicity of a non-anticoagulant, but
antithrombotic algal heterofucan from the Spatoglossum schröederi in
Wistar rats
Periódico: Brazilian Journal of Pharmacognosy
Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 21(4): Jul./Aug. 2011
Fator de impacto: 0.68
ISSN: 0102-695X (Printed version)
ISSN: 1981-528X (Online version)
Qualis: Medicina II – B3
31
Almeida-Lima J. PPGCSA/CCS
Indexada: SCOPUS, SciELO, EMBASE e GEOBASE
5.4. Artigo 3
Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-
inhibition effects of a non-anticoagulant, but antithrombotic algal
heterofucan
Periódico: Journal of applied toxicology : JAT.
J Appl Toxicol. 2010 Oct;30(7):708-15. doi: 10.1002/jat.1547.
Fator de impacto: 2.597
ISSN: 0260-437X (Printed version)
ISSN: 1099-1263 (Online version)
Qualis: Medicina II – B1
Indexada: PubMed – indexado por MEDLINE
32
5.1. ARTIGO 1 (SUBMETIDO)
Mar. Drugs 2014, 12, 1-x manuscripts; doi:10.3390/md120x000x
marine drugs ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Evaluation of potential antitumor activity of fucan
nanogel
Jailma Almeida-Lima1,2
, Arthur Anthunes Jacome Vidal1, Dayanne Lopes
Gomes1,2
, Ruth Medeiros Oliveira1, Leonardo Thiago Duarte Barreto Nobre
3,
Mariana Santana Santos Pereira Costa1, Nednaldo Dantas-Santos
2, Helena
Bonciani Nader3, Francisco Miguel Gama
4, Edda Lisboa Leite
1, Hugo Alexandre
Oliveira Rocha1,2*
1
Laboratory of Biotechnology of Natural Polymers (BIOPOL), Department of
Biochemistry, Federal University of Rio Grande do Norte (UFRN), Natal-RN 59078-
970, Brazil; E-Mails: [email protected] (J.A.-L.); [email protected]
(A.A.J.V.); [email protected] (D.L.G.); [email protected]
(R.M.O.); [email protected] (M.S.S.P.C); [email protected] (E.L.L);
[email protected] (H.A.O.R) 2
Graduate Program in Health Sciences, Federal University of Rio Grande do Norte
(UFRN), Natal-RN 59078-970, Brazil; E-Mails: [email protected] (N.D.-S.) 3
Department of Molecular Biology, Department of Biochemistry, Federal University
of São Paulo - UNIFESP, São Paulo-SP, 04044-020, Brazil; E-Mails:
[email protected] (L.T.D.B.N); [email protected] (H.B.N) 4 IBB—Institute for Biotechnology and Bioengineering, Centre for Biological
Engineering, Minho University, Braga 4704-553, Portugal; E-Mails:
[email protected] (F.M.G.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +55-84-3215-3416 (ext. 207); Fax: +55-84-3211-9208.
Received: / Accepted: / Published:
OPEN ACCESS
33
Abstract: Fucan is a term that defines a family of homo- and
heteropolysaccharides containing sulfated L-Fucose. In this work a
heterofucan (Fucan A) from seaweed Spatoglossum schröederi was
thiolated and treated by ultrasonication, giving rise to intermolecular
disulfide bonds and to the formation of fucan A nanogels. Fucan A nanogel
decorated with polyethylene glycol (FucA:PEG15) was stable for over one
month and showed an average diameter of 187 ± 11 nm in aqueous solution
and a zeta potential of -23.1 ± 2.0 mV, as measured by dynamic light
scattering. This nanogel inhibits the proliferation of 786-0 renal
adenocarcinoma cells in a dose-dependent way. Flow cytometric analysis
showed that FucA:PEG15 induces apoptosis through caspase and caspase-
independent mechanisms. The nanogel labeled with FITC was completely
taken up by 786-0 cells after 1 hour. When we stopped the cell endocytosis,
the FucA:PEG15 antiproliferative effect was abolished. In additon,
FucA:PEG15 has antioxidant activity and also exhibits antiangiogenic
activity . These data show that fucan A nanogel exhibits several activities
(antiproliferative, antioxidant, and antiangiogenic) and therefore its potential
for cancer therapy should be further investigated.
Keywords: fucan; sulfated polysaccharide; brown seaweed; nanogel;
cytotoxicity
1. Introduction
The term cancer is used to refer to more than one hundred different types of diseases
that have in common the characteristic uncontrolled proliferation of anaplastic cells.
These cells, at some point, will invade other tissues and organs. It is estimated that more
than 21 million people will contract cancer and 13 million deaths are expected by 2030.
Although cancer accounts for around 13% of all deaths in the world, more than 30%
could be prevented by modifying or avoiding key risk factors [1].
The use of chemotherapy for the treatment of cancer raises concerns about the
debilitating side effects of this form of treatment; on the other hand, poor cellular
internalization and insufficient intracellular drug release reduces its efficacy [2].
The development of nanomedicine aims to solve the issues associated with the
systemic administration of toxic pharmaceuticals. Thus, chemotherapeutic agents have
been encapsulated, conjugated, entrapped, or loaded into nanoformulations, resulting in
the site-specific drug delivery, thereby reducing the systemic toxicity [3].
Among the available nanosystems, nanogels are particularly attractive since they are
easy to produce, are affordable, and may effectively incorporate a variety of drugs,
including biopharmaceuticals [4]. Nanogels are composed of cross-linked three-
dimensional polymer chain networks that are formed via covalent linkages or self-
34
assembly processes. An increased interest has been witnessed recently for smart
nanogels allowing site-specific and controlled drug release, paving the way for the
development of improved cancer therapeutic formulations [5].
Among the numerous polymers that have been proposed for the preparation of
nanogels, polysaccharides have a number of advantages over the synthetic polymers,
which were initially employed in the field of pharmaceutics [6]. Fucans are
polysaccharides containing substantial amounts of L-fucose and sulfate ester groups,
expressed by brown seaweed [7] and some marine invertebrates (sea urchins and sea
cucumbers) [8]. Algal fucans showed several biological/pharmacological activities such
as antithrombotic, antiviral, anticoagulant, antioxidant, and anti-inflammatory
[9,10,11,12]. In addition, fucan anti-cancer activities have been reported frequently in
recent years, and the potential mechanisms of action were also investigated
[13,14,15,16].
Among the seaweed fucans, we can highlight those extracted from Spatoglossum
schröederi (Dictyotaceae). This brown seaweed is found along almost the entire
Brazilian coast (about 8000 km). The permanent availability of this organism in large
amounts makes it an excellent choice for prospecting bioactive compounds. It
synthesizes three fucans and the one obtained in the larger quantity was named fucan A.
We showed that fucan A is not toxic in vivo [17], but it demonstrated antiproliferative
activity against several tumor cell lines [18]. However, this activity was observed only
for high concentrations, probably because fucan A, due its ionic nature, binds onto a
great variety of proteins, becoming unavailable to perform the bioactivity of interest.
Another kind of sulfated polysaccharide known as heparin binds several proteins [19]
including extracellular matrix proteins [20]. In order to diminish this inactivating
effect, Bae and colleagues synthesized a heparin nanogel cross-linked with disulfide
linkages. When B16-F10 tumor cells were treated with heparin nanogel, their
proliferation was inhibited by about 50%, whereas free heparin alone inhibited the cell
growth to a much smaller extent (~10%) [21].
Previously, we chemically modified fucan A by grafting hexadecylamine to the
hydrophilic backbone. The obtained amphiphilic material self-assembled into fucan A
nanogel, which showed antiproliferative activity against human renal tumor cells (786-0
cells) [22]. In this study fucan A was covalently linked to thiol groups and then cross-
linked with disulfide linkages to produce fucan A nanogel for efficient cellular uptake.
This way, a fully hydrophilic nanogel is obtained. In addition, we investigated the
antiproliferative effect of the newly developed fucan A nanogels using 786 tumor cell
lines. The data obtained indicated that the fucan A nanogel exhibits high stability and is
a more powerful inhibitor of cell growth than free fucan A.
2. Results and Discussion
2.1. Synthesis of the fucan A nanogels
In the present work, fucan A was chemically modified with thiol groups and then
cross-linked with disulfide linkages to produce reducible fucan A nanogels. For this
purpose, the carboxyl groups of fucan glucuronic acids were oxidized and conjugated to
35
cysteamine by reductive amination, yielding thiolated fucan A. Then, the nanogels
were synthesized by a method that comprises two steps: Thiolated fucan was initially
co-dissolved in DMSO with PEG, enabling the interaction between the two polymers,
presumably by hydrogen bonding, resulting in the spontaneous formation of nanosized
complex particles in the DMSO phase. In the second step, thiolated fucan A monomers
within the inner core of the complexes were covalently linked together by disulfide
linkages. This was achieved by ultrasonic treatment, which promotes the formation of
free radicals, which in turn accelerate the oxidation reaction of the thiol groups of the
cysteamine linked to fucan A. The crosslinked fucan nanogels were finally obtained by
withdrawing the DMSO and free PEG through exhaustive dialysis (Figure 1A).
Figure 1. A) Scheme of the synthesis of fucan A nanogels. B)
Transmittance of solution containing nanogels of fucan A prepared using
various weight ratios of PEG (0, 2.5, 5.0, 10, 15, 30) at 400 nm.
The effect of the fucan:PEG ratio on the size of nanogels was investigated. The fucan
A concentration was kept constant and PEG content increased at the ratio of 2.5, 5.0,
10, 15, and 30 times the concentration of fucan A; hence, the obtained samples were
called FucA:PEG2.5, FucA:PEG5.0, FucA:PEG10, FucA:PEG15, and FucA:PEG30,
respectively. We can observe (Figure 1B) that in the absence of PEG the transmittance
value was kept at 100%, i.e., similar to the control of dissolved fucan A. However, with
increasing PEG content, the transmittance gradually decreases to about 60%, for the
higher ratio of FucA:PEG 30, indicating the formation of colloidal particles, the
nanogels, as shown in the following section.
36
2.2. Characterization of the nanogels
The average hydrodynamic diameters of nanogels were measured at a concentration
of 1 mg/mL in water by dynamic light scattering (DLS). The effect of various fucan
A:PEG weight ratios on nanogel size, polydispersity (PDI), and zeta potential was
analyzed. As we can see in Table 1, a weight ratio of FucA:PEG of 30, originates a very
large nanogel (917.18 ± 83.12 nm), whereas the others showed a size ranging from
186.95 ± 10.62 to 301.44 ± 2.26. Bae and colleagues [21], using the same method to
obtain heparin nanogels, elected a weight ratio of heparin:PEG of 15 as the best one
because it would favor stronger interactions between PEG and heparin, leading to the
formation of compact nanogels. This observation is in agreement with ours, since
FucA:PEG15 showed the smaller size (186.95 ± 10.62 nm) among the tested
combinations. However, the size of FucA:PEG15 colloidal micelles is in the same order
of magnitude of other nanogels obtained with fucan A using a different synthetic route
[22], and of others obtained using different acidic polysaccharides and the same
synthesis approach. For instance, heparin nanogels resulted in a stable structure with an
average diameter of 248.7 ± 26.8 nm [21] and acetylated hyaluronic acid gave rise to
nanogels ranging from 275 ± 4 to 447 ± 8 nm [23].
The size of nanomaterials is an extremely important factor determining its fate in
vivo—biodistribution and pharmacokinetics—since it affects namely the phagocytosis
and ability to cross biological barriers. According to Dong and Mumper [24],
nanoparticles with a size around 220 nm are ideal targets for passive targeting of tumors
since the majority of solid tumors exhibit vascular pore cut-offs between 380 and 780
nm. Thus, using the size as a parameter of choice, all fucan A nanogels, except
FucA:PEG30, have potential application against tumor cells. However, other parameters
should be evaluated to confirm this statement.
Table 1. Physicochemical characteristics of nanogels obtained using
different ratios of the polyethylene glycol (PEG) determined by DLS.
Samples Diameter
(nm)
Polydispersity
(PDI)
Conductance
(µS)
Zeta
Potencial
(mV)
FucA:PEG2.5 301.44 ± 2.26 0.69 ± 0.03 193 -28.12 ± 1.55
FucA:PEG5.0 297.15 ± 30.97 0.48 ± 0.09 254 -28.61 ± 0.15
FucA:PEG10 277.10 ± 17.41 0.49 ± 0.03 548 -26.11 ± 0.05
FucA:PEG15 186.95 ± 10.62 0.54 ± 0.06 347 -23.08 ± 1.96
FucA:PEG30 917.18 ± 83.12 0.84 ± 0.09 339 -22.18 ± 0.13
The zeta potential was negative for all nanogels, ranging from -22.18 ± 0.13 to -
28.61 ± 0.15 mV. These results can be explained by the negative charge of fucan A due
to the presence of ionized carboxyl and sulfated groups. This is in good agreement with
37
the zeta potential values found in previous studies for nanoparticles prepared with other
fucans [25], and with chitosan with a blend dextran sulfate [26].
Furthermore, nanogel, as evidenced by the polydispersity index (PDI), ranged
between 0.48 ± 0.09 to 0.84 ± 0.09. The PDI is dimensionless and scaled such that
values smaller than 0.05 are rarely seen other than with highly monodisperse standards.
Values greater than 0.7 are indicative of samples with a very broad size distribution and
probably not suitable for analysis using the dynamic light scattering (DLS) technique
[27,28]. Thus, the best nanogels regarding the heterogeneity of size distribution were
FucA:PEG5.0, FucA:PEG10, and FucA:PEG15.
Size and surface morphology of fucan A nanogels were evaluated by SEM. Figure 2
shows that not all formulations presented a spherical shape, in the case of FucA:PEG2.5
nanoparticles were not detected, while in the case of FucA:PEG30 particles with a
fibrous shape were observed. It can be speculated that some phase transition may have
occurred during the drying of the samples, since in the case of FucA:PEG2.5 nanosized
particles were detected by DLS. Nonetheless, these results demonstrate the critical
relevance of the balance of fucan A vs PEG, suggesting that the arrangement of the two
polymers determines the organization of the nanogel. On the other hand, FucA:PEG5.0,
FucA:PEG10, and FucA:PEG15 showed a spherical shape with an average diameter of
113.33 ± 0.23 nm, 101.85 ± 0.43 nm, and 98.25 ± 1.69 nm, respectively (Figure 2F).
The diameter measured by DLS was slightly larger than the one obtained from SEM,
presumably due to the swelling of fucan A nanogels in water (taking into account that
carbohydrate polymers are highly hygroscopic), since the samples needed to be dried
for analysis by SEM, as noted by other authors [21,23].
Figure 2. SEM photograph of nanogels with various weight ratios of
fucan A:PEG (A) 2.5, (B) 5, (C) 10, (D) 15, (E) 30, and F) average
diameter of the nanogels. The diameters of particles in SEM images were
measured by comparing them with the size bar.
38
2.3. FTIR of the fucan A nanogels
FTIR spectra of fucan A, FucA:PEG10, and FucA:PEG15 are depicted in Figure 3.
Characteristic sulfate absorptions were identified in the FTIR spectra: bands around
1265 cm−1
for asymmetric S=O stretching vibration and around 1041 cm−1
for
symmetric C–O vibration associated with a C–O–SO3 group [18]. The bands at 813–850
were caused by the bending vibration of C–O–S. At 3200–3500 cm−1
, fucan A and
fucan A nanogels showed bands from the stretching vibration of O–H [9]. The band
around 2900 cm−1
corresponds to stretching vibrations of CH2, which is higher in fucan
nanogel spectra due to the presence of stretching vibrations of CH2 in cysteamine
residues [29], as further confirmed by the band at 1468 cm−1
, featuring a higher
intensity in fucan nanogel spectra. This band corresponds to C–H symmetric
deformation vibration [30]. A band around 1410 cm−1
was identified in all spectra and
was assigned to symmetric vibration of COO− of glucuronic acid. The presence of
glucuronic acid was also confirmed by the antisymmetric stretching vibration of COO−
at 1618 cm−1
[31], which overlaps with the vibration of water. The H2O molecule has
strong IR absorbance with three prominent bands around 3400 (O–H stretching), 2151
(water association), and 1618 cm−1
(H-O-H bending) [32] in the fucan A spectrum.
However, the band intensity decreases in fucan nanogels. The absorption band due to S–
H stretching vibrations of the thiol group was also observed at 2600–2550 cm−1
[33].
Figure 3. Infrared of fucan A and nanogels FucA:PEG10, FucA:PEG15,
Fuc:PEG30.
2.4. Nanogel Stability
The nanogels FucA:PEG10 and FucA:PEG15 showed the smallest particle size and
best morphological features, and therefore were chosen for further characterization. In
order to investigate the stability of the macromolecular association over time, we used
39
DLS. As presented in Figure 4, the fucan A nanogels were highly stable in aqueous
solution, suggesting that their structural integrity was preserved.
Figure 4. Stability of fucan A nanogels FucA:PEG10 and FucA:PEG15
accessed by DLS. The nanogels were stored at 4 °C for up to 42 days and
analyzed in DLS at temperature 25°C.
2.5. Cytotoxicity assay
The cytotoxic of FucA:PEG10, FucA:PEG15, and Fucan A upon 786-0 cells was
investigated for 24 h using a colorimetric MTT-based assay (Figure 5). Fucan A
displayed a dose-dependent inhibitory effect, which was quite expressive (by about 3
fold) in the case of nanogels. FucA:PEG10 and FucA:PEG15 also showed a dose-
dependent effect, reaching saturation at around 0.04 and 0.06 mg/mL respectively. In
addition, the data also indicate that FucA:PEG15 is slightly more efficient as an
antiproliferative compound than FucA:PEG10.
Dantas-Santos et al. [22], using fucan A nanogels grafting hexadecylamine,
evaluated the cell viability of various types of tumor cells. The 786-0 cell line was the
more susceptible one (inhibition of ~40%). However, this inhibition was obtained only
for a concentration as high as 500 µg/mL, ten times higher than the required using
FucA:PEG15.
40
Figure 5. Inhibition of proliferation of 786 cells incubated with Fucan A,
FucA:PEG10, or FucA:PEG15 nanogels at various concentrations for 24
hours. Data are expressed as means ± standard deviation. a,b,c,d,e The
different letters indicate significant difference between the concentrations
of the same compound (p < 0.05).
2.6. Apoptotic effect of fucan A nanogels
Since FucA:PEG15 was the most potent cell growth inhibitor, it was chosen for
mechanistic studies, namely to determine whether cell death for apoptosis was
responsible for the observed effect. For this purpose, 786-0 cells were treated with
FucA:PEG15 (64 µg/mL) for 24 hours, followed by flow cytometric analysis.
Cell apoptosis features the exposure of phosphatidylserine on the external side of the
cell membrane, which can be recognized by annexin V. On the other hand, necrotic
cells can be identified using propidium iodide (PI), which stains only necrotic cells
bearing a compromised, porous cell membrane. Generally, cells stained with annexin V
are indicative of early apotosis and stained cells with PI, indicative of necrosis, while
double labeling is indicative of late apoptosis [34].
In Figure 6 we can see the results of flow cytometry analysis of cells cultivated in the
presence and absence of nanogels (control group). For the control group (Figure 6A),
91.4% of the cells are negative for annexin V and PI. However, after the FucA:PEG15
treatment, this number dropped to 59.0%, whereas the percentage of cells stained with
annexin V increased from 7.8 to 39%. Furthermore, the percentage of cells stained with
PI did not change (Figure 6B). These data indicate that FucA:PEG15 inhibits
proliferation by inducing apoptosis.
41
In order to determine the role of caspases in the FucA:PEG15 nanogel-induced
apoptosis, 786-0 cells were incubated with ZVAD-FMK. As can be seen in Figure 6B,
in the presence of this pan-caspase inhibitor, the percentage of cells positive for annexin
decrease from 39 to 27%. Additionally, in the presence of E64 (cysteine peptidase
inhibitors, mainly cathepsin and calpains specific) the effect also decreased to about
10% of inhibition (Figure 6D). These data indicate that FucA:PEG15 has a complex
mechanism of apoptosis induction.
Several fucans have been shown to induce apoptosis in different types of tumor cells,
an event often related to caspase activation [35,36,37]; however, other proteins involved
in cell survival pathways may be affected by the presence of fucans in the culture
medium, such as proteins from ERK1/2MAPK pathway [16,38] and PI3K/AKT
pathway [13], apoptosis-inducing factor (AIF) [10], JNK/c-Jun/AP-1 pathways, and
death receptor-mediated and mitochondria-mediated apoptotic pathways [14].
The analysis of the aforementioned data suggests that the mechanism of apoptosis
induction by fucans is very complex, in agreement with the results obtained in this work
using the fucan A nanogel. Further work will be dedicated to identifying mainly the cell
proteins involved in the FucA:PEG15 mechanism of action to induce apoptosis.
Figure 6. Cytometry with cells 786-0. A) Control (Anexin + PI); B)
FucA:PEG15; C) FucA:PEG15 + ZVAD and D) FucA:PEG15 +E64 all
with the same concentration (64 µg/mL).
42
2.7. FucA:PEG15 intracellular uptake
The intracellular uptake of nanogels was investigated by confocal microscopy and
flow cytometry. For this purpose, the nanogel was conjugated with FITC and used as
fluorescence probe, allowing the observation of its interaction with the 786-0 cells.
The antiproliferative effect (around 40% for a concentration of 0.06 mg/mL) was not
affected by the FITC labeling. It is important to mention that FITC alone has no activity
on 786-0 cell proliferation (data not shown). The confocal observation of 786-0 cells
after incubation with FucA:PEG15+FITC for 1 h allowed the detection of internalized
fucan nanogel (Figure 7B). In addition, the nanogel is observed in the perinuclear space
(Figure 7C). The free FITC was also assayed under the same conditions and did not
show any binding to cells or extracellular matrix (data not shown).
The internalization kinetics was analyzed by flow cytometry (Figure 7). As can be
seen in Figure 7D, 15 min suffice for the cells to become labeled with
FucA:PEG15+FITC. Furthermore, the incubation of the cells in the presence of an
excess of fucan A (ten times more) prevented the entry of the nanogel into the cell,
probably because the same cell receptor/endocytic pathway was used.
Lira and colleagues [25] assigned a similar antiproliferative activity against J774
macrophages and NIH-3T3 fibroblasts to fucan nanoparticles. These authors also
showed that the fucan nanogels need to be internalized in order to exert their
antiproliferative activity. In order to confirm this hypothesis, the cells were exposed to
FucA:PEG15 nanogels (from 0.01 to 0.1 mg/mL) for 2 hours at 4 °C (condition in
which the endocytosis is substantially inhibited). After washing and addition of medium
without nanogel, the cell proliferation was measured after 24 hours. In another set of
experiments the 786-0 cells were incubated with FucA:PEG15 nanogels (from 0.01 to
0.1 mg/mL) for 6, 12, 18, and 24 hours at 4 °C. In both set of experiments,
FucA:PEG15 nanogels did not demonstrate an antiproliferative effect (data not shown).
Figure 7. Confocal microscopy and flow cytometry of 786-0 cells treated
with nanogel or Fucan A. A) nuclei stained with DAPI; B) region of
cytoplasm stained with FITC and C) overlap of the images A and B. The
cells used for confocal microscopy were treated only with FucA:PEG15
nanogels. Flow cytometry D) FucA:PEG15 at the same concentration
(100 µg/mL) at different times 15, 30 min, and 1h; E) FucA:PEG15
(1000 µg/mL), FucA:PEG15+FITC (100 µg/mL) + Fucan A (1000
µg/mL) and FucA:PEG15 (100 µg/mL).
43
2.8. Antioxidant activity of the nanogel
Antioxidants are substances that are useful for fighting cancer and other processes
that potentially lead to various diseases; antioxidants act by preventing the onset of
cancer during carcinogenesis, and they are generally beneficial to cells [39].
In this work, the antioxidant activity was evaluated in different assays: reducing
power, DPPH radical-scavenging assay, superoxide radicals, and total antioxidant
capacity (TCA). The results are shown in Table 2. The FucA:PEG15 nanogel and fucan
A did not show antioxidant activity in power reducing, superoxide anion scavenging,
and DPPH radical-scavenging assays. However, in the total antioxidant activity assay,
both the nanogel and fucan A were positive.
Table 2. Antioxidants activities with nanogel and fucan A.
Antioxidant activity Fucan A FucA:PEG15
Reducing power (%) nd nd
DPPH (%) nd nd
Superoxide radical (%) 0.0 1.6
TCA (mg/g ascorbic acid equivalents) 26.20a 36.31
b
nd - not detected. a,b Different letters indicate significant difference between
the samples of the same concentration (p < 0.05).
44
2.9. Anticoagulant activity of the nanogel
Fucans from various seaweeds, including different Dictyotales [31,40], possess
anticoagulant activity. In this way, we evaluated the anticoagulant activity of
FucA:PEG15 using the PT and aPTT test. But in all conditions evaluated (from 10 to
100 µg/mL), FucA:PEG15 showed no anticoagulant activity (data not shown).
2.10. FucA:PEG15 inhibits the angiogenesis
Using the matrigel assay, we investigated whether FucA:PEG15 can inhibit
angiogenesis in vitro. This matrigel is composed mainly of laminin, TGF-β, and
entactin, and has been used to study angiogenesis in vivo and in vitro [41]. As shown in
Figure 8 (B, C, and D), the nanogels inhibit the formation of capillary-like tubes by
endothelial cells. The FucA:PEG15 significantly inhibited capillary tube formation with
increasing concentration of nanogel, 12.5 µg/mL (Figure 8B) to 100 µg/mL (Figure
8D), compared to the control group (Figure 8A). In addition, the nanogels did not show
any cytotoxic effect against RAEC cells, even when they were incubated on the matrigel
(data not shown).
Angiogenesis is one of the most important events for the maintenance and growth of
tumors. The newly formed vessels are responsible for delivering oxygen and nutrients to
the growing tumor. Various drugs have been developed for use as antiangiogenic agents
and thus combat the development of tumors [42]. The fucan nanogels studied here
exhibit cytotoxic activity against tumor cells, antioxidant activity, and antiangiogenic
activity, properties that have been extensively researched in compounds used in the
treatment of tumors. In addition, nanogels here are particles with potential for drug
carriers, including anticancer.
45
Figure 8. Effects of nanogel on capillary tube formation of rabbit aortic
endothelial cells (RAEC). The cells were non-treated (A-control group)
and treated with different concentrations of FucA:PEG15 (B- 12.5
μg/mL, C- 50 μg/mL, and D- 100 μg/mL). 50x magnification.
3. Experimental Section
3.1. Materials
Poly (ethylene glycol (PEG, Mw 4000)), MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-
diphenyltetrazoliumbromide), and cellulose acetate dialysis bags, with 6,000 or 12,000
MWCO, were purchased from Sigma Chemical Company (St. Louis, MO, USA). L-
glutamine, sodium bicarbonate, penicillin, streptomycin, sodium pyruvate, and
phosphate buffered saline (PBS) were purchased from Invitrogen Corporation
(Burlington, ON, USA). The 786-0 renal adenocarcinoma (ATCC CRL-1932) and
RAW 264 monocyte macrophage (ATCC TIB-71) cell lines were donated by Dr.
Carmen Ferreira (Department of Biochemistry, UNICAMP, Brazil). Cell culture
medium components (RPMI-1640 Medium or DMEM), trypsin, and fetal calf serum
(FCS) were obtained from Cultilab (Campinas, SP, Brazil). Rabbit aortic endothelial
cells (RAEC) were maintained at 37 °C under stress of 2.5% CO2, in HAM-F12
medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine
serum. All other solvents and chemicals were of analytical grade. The fucan A from
46
Spatoglossum schröederi was obtained as described by Almeida-Lima and colleagues
[18].
3.2. Synthesis of fucan A nanogels
Fucan A (10 mg, 50 µmol) dissolved in 0.1 M citrate buffer (pH 3.0) was reacted
with sodium periodate (112 mg, 523 µmol) for 2 h at 4 ºC. After dialysis against
distilled water (Mw cutoff of 6 kDa), the oxidized fucan A was reacted for 2 h with
cysteamine hydrochloride (60 mg, 523 µmol) in 0.1 M phosphate-buffered saline (PBS,
pH 7.0). Then, 10 ml of 0.1 M NaBH4 solution was slowly added, and the mixture was
stirred for 1 h at 4 ºC. The solution was dialyzed and then lyophilized. Thiolated fucan
A was solubilized in DMSO. Five milligrams of thiolated fucan was mixed with PEG in
distilled water at different weight ratios of PEG to thiolated fucan A, and then
lyophilized. The dried mixture of thiolated fucan A and PEG was solubilized in 10 mL
of DMSO with incubation for 4 h at 37 ºC. The solution was sonicated for 3 min using a
Sonicator Sonics Vibra-Cell (20 kHz, output control 3) to facilitate the oxidation
reaction of thiol groups. The resultant fucan A nanogels were purified by extensive
dialysis against distilled water (Mw cutoff of 12 kDa). The degree of thiolation was
estimated as approximately 24%, by comparing the relative peak intensity ratio found in
2600–2550 cm−1
at nanogels and fucan A spectrum, which correspond to S–H stretching
vibrations of the thiol group, as shown in the results section.
3.3. Transmittance of hydrogels
The transmittance of the solution containing nanogels of fucan A prepared using
various weight ratios of PEG (0, 2.5, 5.0, 10, 15, 30) was measured at 400 nm with a
Multiskan Ascent Microplate Reader (Thermo Labsystems, Franklin, MA, USA)
3.4. Dynamic light scattering (DLS)
The effective hydrodynamic diameter and zeta-potential of nanozymes was measured
by photon correlation spectroscopy using Brookhaven 90 Plus Nanoparticle Size
Analyzer (Brookhaven Instruments Corporation, New York, USA). Fucan A nanogel
dispersions were prepared in water at 25 °C at a concentration of 1 mg/ml. The zeta
potential was obtained by using a disposable capillary cell in automatic mode on the
same instrument.
3.5. Scanning electron microscopy (SEM)
Size and shape of the fucan A nanogels were evaluated by scanning electron
microscopy (SEM). For both experiments, 50 µL of the nanogel solution was deposited
onto a clean mica surface, allowed to dry at 25 ºC, and then observed in a Shimadzu
electron microscope, model SSX550 (Shimadzu Scientific Equipment, UK).
47
3.6. Fourier Transform Spectra (FT-IR)
All samples (5mg) were mixed thoroughly with dry potassium bromide. A pellet was
prepared, and the infrared spectrum was measured on a Thermo Nicolet Nexus
spectrometer instrument (model Nexus 470 FT-IR).
3.7. Stability
The stability was evaluated analyzing the nanogel, solubilized in water, for its size
distribution, as described above. The analysis was performed weekly, up to 42 days, as
described above. The nanogel solution was kept at 4 °C during the study and removed
for analysis about 24 hours before each measurement, always performed at 25 °C.
3.8. MTT Cytotoxicity assay
The cells were grown in 25 cm2 flasks in DMEM medium. For the experiments, the
cells were seeded into 96-well plates at a density of 5 × 103 cell/well and allowed to
attach overnight in 100 μL medium. The fucan A nanogels and the non-modified
polysaccharide were added at a final concentration of 8.0, 16, 32, 48 and 64 μg/mL, for
24, 48, and 72 h at 37 ºC and 5% CO2. In some tests, the RAEC cells were grown on
matrigel in 24-well plate at a density of 5 × 104 cell/well in 500 μL medium.
After incubation, traces of samples were removed by washing the cells twice with
100 μL PBS; then, MTT (1mg/mL) dissolved in 100 μL of fresh medium was added and
incubated for 4 h at 37 ºC, 5% CO2. The medium was aspirated, and the MTT-formazan
product was dissolved in 100 μL of ethanol and estimated by measuring the absorbance
at 570 nm in a Multiskan Ascent Microplate Reader (Thermo Labsystems, Franklin,
MA, USA). All concentrations were tested in triplicates and the experiment was
repeated at least three times. The percentage of cell proliferation inhibition was
calculated as follows:
%Inhibition =
3.9. Conjugation of fucan with fluorescein
In order to visualize cellular uptake, fluorescein-labeled fucan A and the nanogel
(FucA:PEG15) were prepared by conjugating fluorescein sodium salt (Sigma). In brief,
5 mg of fucan A or nanogel were reacted with 1 mg of fluorescein in 0.1 M PBS
solution (pH 7.0) and stirred for 1 hour protected from light, at room temperature. The
solution was dialyzed against deionized water (Mw cutoff of 12 kDa) and then
lyophilized.
48
3.10. Analysis by Flow Citometry
The number of apoptotic cell deaths induced by FucA:PEG15 nanogels was
measured by flow cytometry using Annexin V-FITC Apoptosis Detection Kit (BD
Biosciences, San Diego, CA, USA). The 786-0 cells were placed in a 6-well plate (2 ×
105 cells/mL) and after 48 h stimulated to enter G0 using a medium without serum for
24 h. Next, the medium was replaced by DMEM supplemented with 10% FBS, in the
presence of fucan A (64 µg/mL) or FucA:PEG15 (64 µg/mL). A negative control was
prepared without the presence of polysaccharide. The cell effect of FucA:PEG15
incubated with pan-caspase inhibitor ZVAD-FMK (carbobenzóxy valyl-alanyl-aspartyl-
[O-methyl]-fluorometilcetone) or the inhibitor cysteine proteinase (E-64) was also
tested. After 24 h the cells were harvested, and after centrifugation the cell pellets were
washed twice with cold PBS and suspended in 50 μL of 1 × Anexinn-V buffer. Cells
were then incubated with 5 μL of annexin V-FITC and 2 μL of PI at room temperature
for 15 min in the dark. After incubation, 300 μL of 1 × binding buffer (10 mM
HEPES/NaOH, 140 m M NaCl, 2.5 mM CaCl2, pH 7.4) was added to each tube. The
cells were immediately analyzed by Facscanto II flow cytometry (BD Biosciences, San
Diego, CA, USA) in FL1 channel (excitation at 488 nm and emission at 530 nm) and
FL3 (excitation at 650 and emission at 630 nm). A total of 40.000 events were acquired.
For data analysis, FlowJo® Analysis Software version 9.3.2 was used.
3.11. Confocal analysis
The 786-0 cells (1 × 105) were placed on 12 mm-diameter glass cover slips in 24-
well cluster plates (Nunc; Naperville, IL, USA). After 3 days in culture, the cells were
washed three times with PBS (0.1 M pH 7.4), and then treated with the fluorescein-
labeled fucan A or nanogel FucA:PEG15 (64 μg/mL) in serum-free RPMI medium for
15, 30, or 60 min at 37 °C. Afterwards, the cells were washed and fixed with 2%
paraformaldehyde (4 °C). Then, after washing with PBS, the cell nuclei were stained
with DAPI solution (50 μg/mL in PBS) for 30 min and washed five times in PBS, once
in water, and glass cover was mounted in Fluoromount-G (E.M. Sciences; Ft.
Washington, WA, USA) and examined with a confocal or fluorescence microscope
(Zeiss Axio Examiner LSM 710, Jena, Germany).
3.12. Antioxidant activity
Four assays were performed to analyze the antioxidant activity of the fucan A and
nanogel obtained: reducing power, DPPH, superoxide radical scavenging, and total
antioxidant capacity as previously described [43,44].
3.13. Anticoagulant activity
49
Both the protrombin time (PT) and activated partial thromboplastin time (aPTT)
coagulation assays were performed with a coagulometer as described earlier [43] and
measured using citrate-treated normal human plasma. All assays were performed in
duplicate and repeated at least three times on different days (n = 6).
3.14. Matrigel tube formation assay
The tube formation assay was adapted from Dreyfuss et al. [45]. Matrigel purified
from EHS tumor was thawed at 4 °C on ice and plated on the bottom of 24 well-plates,
and incubated at 37 °C for 16 h for gelification. ECs (105 cells) were seeded on Matrigel
in F12 medium containing 10% FBS and different amounts (12.5, 25, 50, and 100
µg/mL) of FucA:PEG15 nanogel or saline (control). The cultures were maintained at 37
°C in a 2.5% CO2 humidified atmosphere for 24 h. Each treatment was performed in
triplicate. Tube formation was examined under an inverted light microscope at 50x
magnification. Three images were randomly taken in different areas.
3.15. Statistical analysis
All data are presented as the average ± SEM. Tests for significant differences
between the groups were done using one-way ANOVA with multiple comparisons
(Kruskal-Wallis) using GraphPad Prism 4.0 (GraphPad software, San Diego, EUA).
4. Conclusions
Several polysaccharide-derived nanoparticles have been reported as inert vehicles
used to carry drugs or other molecules. Here we synthesize a fucan-derived nanogel
(FucA:PEG15), which showed cancer-specific antiproliferative activity, antioxidant and
angiogenic. All these activities are indicated as being important for anti-cancer drugs.
Thus, FucA:PEG15 is therefore more than an inert vehicle and its activity could be
potentially applied for cancer cell treatment associated with various anti-cancer drugs.
Further work is being carried out on the incorporation of drugs into the FucA:PEG15, as
well as on the mechanisms of its antiproliferative activity. These results will be reported
in the near future.
Acknowledgments
Research was supported by the Ministério de Ciência, Tecnologia e Informação
(MCTI), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Fundação de Apoio a Pesquisa do Estado do Rio Grande do Norte (FAPERN),
Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), and Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. Hugo AO
50
Rocha and Helena B Nader are CNPq fellowship-honored researchers. Ruth M Oliveira,
Leonardo TDB Nobre had a Ph. D. scholarship from CNPq; and Jailma Almeida-Lima
and Nednaldo Dantas-Santos had a Ph. D. scholarship from CAPES. This research was
submitted to the Graduate Program in Health Sciences at the Federal University of Rio
Grande do Norte as part of the D.Sc. thesis of Jailma Almeida-Lima. Miguel Gama
thanks CAPES for support through the program “Ciência sem fronteiras”.
References
1. World Health Organization. 2011. Cancer. Available online:
http://www.who.int/mediacentre/factsheets/fs297/en (accessed on 27 October
2013).
2. Zhou, T.; Xiao, C.; Fan, J.; Chen, S.; Shen, J.; Wu, W.; Zhou, S. A nanogel of on-
site tunable pH-response for efficient anticancer drug delivery. Acta Biomater.
2013, 9, 4546 – 4557.
3. Yallapu, M.M,; Jaggi, M.; Chauhan, S.C. Design and engineering of nanogels for
cancer treatment. Drug Discov. Today 2011, 16, 457–463.
4. Gonçalves, C.; Pereira, P.; Gama, M. Self-Assembled Hydrogel Nanoparticles for
Drug Delivery Applications. Materials 2010, 3, 1420–1460.
5. Madhusudana Rao, K.; Mallikarjuna, B.; Krishna Rao, K.S.; Siraj, S.; Chowdoji
Rao, K.; Subha, M.C. Novel thermo/pH sensitive nanogels composed from poly(N-
vinylcaprolactam) for controlled release of an anticancer drug. Colloids Surf B
Biointerfaces 2013, 102, 891–897.
6. Coviello, T.; Matricardi, P.; Marianecci, C.; Alhaique, F. Polysaccharide hydrogels
for modified release formulations. J Control Release 2007,119, 5–24.
7. Ale, M.T.; Mikkelsen, J.D.; Meyer, A.S. Important determinants for fucoidan
bioactivity: a critical review of structure-function relations and extraction methods
for fucose-containing sulfated polysaccharides from brown seaweeds. Mar Drugs
2011, 9:2106–2130.
8. Pomin VH. Fucanomics and galactanomics: marine distribution, medicinal impact,
conceptions, and challenges. Mar Drugs 2012, 10, 793-811.
9. Barroso, E.M.; Costa, L.S.; Medeiros, V.P.; Cordeiro, S.L.; Costa, M.S.P.; Franco,
C.R.; Nader, H.B.; Leite, E.L.; Rocha, H.A. A non-anticoagulant heterofucan has
antithrombotic activity in vivo. Planta Med. 2008,74, 712–718.
51
10. Costa, L.S.; Fidelis, G.P.; Telles, C.B.; Dantas-Santos, N.; Camara, R.B.; Cordeiro,
S.L.; Costa, M.S.; Almeida-Lima, J.; Melo-Silveira, R.F.; Oliveira, R.M.; et al.
Antioxidant and antiproliferative activities of heterofucans from the seaweed
Sargassum filipendula. Mar Drugs 2011, 9, 952–966.
11. Silva, R.O.; dos Santos, G.M.; Nicolau, L.A.; Lucetti, L.T.; Santana, A.P.; Chaves,
L.S.; Barros, F.C.; Freitas, A.L.; Souza, M.H.; Medeiros, J.V. Sulfated-
polysaccharide fraction from red algae Gracilaria caudata protects mice gut against
ethanol-induced damage. Mar Drugs 2011, 9, 2188–2200.
12. Albuquerque, I.R.; Cordeiro, S.L.; Gomes, D.L.; Dreyfuss, J.L.; Filgueira, L.G.;
Leite, E.L.; Nader, H.B.; Rocha, H.A. Evaluation of anti-nociceptive and anti-
inflammatory activities of a heterofucan from Dictyota menstrualis. Mar Drugs
2013,11, 2722–2740.
13. Hyun, J.H.; Kim, S.C.; Kang, J.I.; Kim, M.K.; Boo, H.J.; Kwon, J.M.; Koh, Y.S.;
Hyun, J.W.; Park, D.B.; Yoo, E.S. et al. Apoptosis inducing activity of fucoidan in
HCT-15 colon carcinoma cells. Biol Pharm Bull 2009, 32, 1760–1764.
14. Kim, E.J.; Park, S.Y.; Lee, J.Y.; Park, J.H. Fucoidan present in brown algae induces
apoptosis of human colon cancer cells. BMC Gastroenterol 2010, 10, 96.
15. Ermakova, S.; Sokolova, R.; Kim, S.M.; Um, B.H.; Isakov, V.; Zvyagintseva, T.
Fucoidans from brown seaweeds Sargassum hornery, Eclonia cava, Costaria
costata: structural characteristics and anticancer activity. Appl Biochem Biotechnol
2011,164, 841–850.
16. Nobre, L.T;. Vidal, A.A.; Almeida-Lima, J.; Oliveira, R.M.; Paredes-Gamero, E.J.;
Medeiros, V.P.; Trindade, E.S.; Franco, C.R.; Nader, H.B.; Rocha, H.A. Fucan
effect on CHO cell proliferation and migration. Carbohydr Polym 2013, 1,224–232.
17. Almeida-Lima, J.; Dantas-Santos, N.; Gomes, D.L.; Cordeiro, S.L.; Sabry, D.A.;
Costa, L.S.; Freitas, M.L.; Silva, N.B.; Moura, C.E.B.; Lemos, T.M.A.M.; et al.
Evaluation of acute and subchronic toxicity of a non-anticoagulant, but
antithrombotic algal heterofucan from the Spatoglossum schröederi in Wistar rats.
Rev Bras Farmacogn 2011, 21, 674–679.
18. Almeida-Lima, J.; Costa, L.S.; Silva, N.B.; Melo-Silveira, R.F.; Silva, F.V.; Felipe,
M.B.; Medeiros, S.R.; Leite, E.L.; Rocha, H.A. Evaluating the possible genotoxic,
mutagenic and tumor cell proliferation-inhibition effects of a non-anticoagulant, but
antithrombotic algal heterofucan. J Appl Toxicol. 2010, 30, 708–715.
52
19. Mulloy, B. The specificity of interactions between proteins and sulfated
polysaccharides. An Acad Bras Cienc 2005, 77, 651–664.
20. Medeiros, V.P.; Paredes-Gamero, E.J.; Monteiro, H.P.; Rocha, H.A.; Trindade,
E.S.; Nader, H.B. Heparin-integrin interaction in endothelial cells: downstream
signaling and heparan sulfate expression. J Cell Physiol 2012, 227, 2740–2749.
21. Bae, K.H.; Mok, H.; Park, T.G. Synthesis, characterization, and intracellular
delivery of reducible heparin nanogels for apoptotic cell death. Biomaterials 2008,
29, 3376–3383.
22. Dantas-Santos, N.; Almeida-Lima, J.; Vidal, A.A.; Gomes, D.L.; Oliveira, R.M.;
Santos Pedrosa, S.; Pereira, P.; Gama, F.M.; Oliveira Rocha, H.A. Antiproliferative
activity of fucan nanogel. Mar Drugs 2012, 10, 2002–2022.
23. Park, W.; Kim, K.S.; Bae, B.C.; Kim, Y.H.; Na, K. Cancer cell specific targeting of
nanogels from acetylated hyaluronic acid with low molecular weight. Eur J Pharm
Sci 2010, 40, 367–375.
24. Dong, X.; Mumper, R.J. Nanomedicinal strategies to treat multidrug-resistant
tumors: current progress. Nanomedicine 2010, 5, 597–615.
25. Lira, M.C.; Santos-Magalhães, N.S.; Nicolas, V.; Marsaud, V.; Silva, M.P.;
Ponchel, G.; Vauthier, C. Cytotoxicity and cellular uptake of newly synthesized
fucoidan-coated nanoparticles. Eur J Pharm Biopharm 2011,79, 162–170.
26. Chen, Y.; Mohanraj, V.J.; Wang, F.; Benson, H.A. Designing chitosan-dextran
sulfate nanoparticles using charge ratios. AAPS PharmSciTech 2007, 8, E98.
27. International Standard ISO13321 Methods for Determination of Particle Size
Distribution Part 8: Photon Correlation Spectroscopy, International Organisation
for Standardisation (ISO) 1996.
28. International Standard ISO22412 Particle Size Analysis – Dynamic Light
Scattering, International Organisation for Standardisation (ISO) 2008.
29. Wang, X.; Wang, J.; Zhang, J.; Zhao, B.; Yao, J.; Wang, Y. Structure-antioxidant
relationships of sulfated galactomannan from guar gum. Int J Biol Macromol 2010,
46, 59–66.
30. Xu, X.; Li, S.; Jia, F.; Liu, P. Synthesis and antimicrobial activity of nano-fumed
sílica derivative with N,N-dimethyl-n-hexadecylamine. Life Sci J 2006, 3, 59–62.
31. Camara, R.B.; Costa, L.S.; Fidelis, G.P.; Nobre, L.T.; Dantas-Santos, N.; Cordeiro,
S.L.; Costa, M.S.; Alves, L.G.; Rocha, HA. Heterofucans from the brown seaweed
53
Canistrocarpus cervicornis with anticoagulant and antioxidant activities. Mar
Drugs 2011, 9, 124–138.
32. Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein
secondary structures. Acta Biochim Biophys Sin (Shanghai) 2007, 39, 549–559.
33. Coates, J.P. A Practical Approach to the Interpretation of Infrared Spectra.
Encyclopedia of Analytical Chemistry. Ed. RA Meyers, United Kingdon:
Chichester, 2000. p. 10815-37.
34 Wang, L.; Zhang, H.; Chen, B.; Xia, G.; Wang, S.; Cheng, J.; Shao, Z.; Gao, C.;
Bao, W.; Tian, L.; et al. Effect of magnetic nanoparticles on apoptosis and cell
cycle induced by wogonin in Raji cells. Int J Nanomedicine 2012, 7, 789–798.
35. Aisa, Y.; Miyakawa, Y.; Nakazato, T.; Shibata, H.; Saito, K.; Ikeda, Y.; Kizaki, M.
Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of
caspase-3 and down-regulation of ERK pathways. Am J Hematol 2005, 78, 7–14.
36. Athukorala, Y.; Ahn, G.N.; Jee, Y.H.; Kim, G.Y.; Kim, S.H.; Ha, J.H.; Kang, J.-S.;
Lee, K.-W.; Jeon, Y.-J. Antiproliferative activity of sulfated polysaccharide isolated
from an enzymatic digest of Ecklonia cava on the U-937 cell line. J Appl Phycol
2009, 21, 307–314.
37. Yamasaki-Miyamoto, Y.; Yamasaki, M,; Tachibana, H.; Yamada, K. Fucoidan
induces apoptosis through activation of caspase-8 on human breast cancer MCF-7
cells. J Agric Food Chem 2009, 57, 8677–8682.
38. Nabeyrat, E.; Jones, G.E.; Fenwick, P.S.; Barnes, P.J.; Donnelly, L.E. Mitogen-
activated protein kinases mediate peroxynitrite-induced cell death in human
bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2003, 284, L1112–
L1120.
39. Gomes de Melo, J.; de Sousa Araújo, T.A.; Thijan Nobre de Almeida e Castro, V.;
Lyra de Vasconcelos Cabral, D.; do Desterro Rodrigues, M.; Carneiro do
Nascimento, S.; Cavalcanti de Amorim, E.L.; de Albuquerque, U.P.
Antiproliferative activity, antioxidant capacity and tannin content in plants of semi-
arid northeastern Brazil. Molecules 2010, 15, 8534–8542.
40. Magalhaes, K.D.; Costa, L.S.; Fidelis, G.P.; Oliveira, R.M.; Nobre, L.T; Dantas-
Santos, N.; Camara, R.B.; Albuquerque, I.R.; Cordeiro, S.L.; Sabry, D.A.; et al.
Anticoagulant, Antioxidant and Antitumor Activities of Heterofucans from the
Seaweed Dictyopteris delicatula. Int J Mol Sci 2011, 12, 3352–3365.
54
41. Kang, K.; Lim, D.H.; Choi, I.H.; Kang, T.; Lee, K.; Moon, E.Y.; Yang, Y.; Lee,
M.S.; Lim, J.S. Vascular tube formation and angiogenesis induced by
polyvinylpyrrolidone-coated silver nanoparticles. Toxicol Lett 2011, 3, 227–234.
42. Hillen, F.; Griffioen, A.W. Tumour vascularization: sprouting angiogenesis and
beyond. Cancer Metastasis Rev 2007, 3–4, 489–502.
43. Costa, L.S.; Fidelis, G.P.; Cordeiro, S.L.; Oliveira, R.M.; Sabry, D.A.; Câmara,
R.B.; Nobre, L.T.; Costa, M.S.; Almeida-Lima, J.; Farias, E.H.; et al. Biological
activities of sulfated polysaccharides from tropical seaweeds. Biomed
Pharmacother 2010, 64, 21–28.
44. Vinayak, R.C.; Sabu, A.S.; Chatterji, A. Bio-prospecting of a few brown seaweeds
for their cytotoxic and antioxidant activities. Evid Based Complement Alternat Med
2011, 2011, 673083.
45. Dreyfuss, J.L.; Regatierim C,V.; Lima, M.A.; Paredes-Gamero, E.J.; Brito, A.S.;
Chavante, S.F.; Belfort, R.Jr.; Farah, M.E.; Nader, H.B. A heparin mimetic isolated
from a marine shrimp suppresses neovascularization. J Thromb Haemost 2010, 8,
1828–1837.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons
Attribution license (http://creativecommons.org/licenses/by/3.0/).
55
5.2. CAPÍTULO DE LIVRO
56
57
58
59
Chapter 6
APPLICATION OF MARINE POLYSACCHARIDES IN
NANOTECHNOLOGY
Raniere Fagundes Melo-Silveira, Jailma Almeida-Lima, and Hugo
Alexandre Oliveira Rocha* Laboratório de Biotecnologia de Polímeros Naturais (BIOPOL), Departamento de Bioquímica,
Universidade Federal do Rio Grande do Norte (UFRN), Natal/RN, Brazil
ABSTRACT
Scientific knowledge in the field of nanotechnology has been expanded rapidly over the past
two decades. The potential application of this new technology in several areas of science has
attracted attention to the development of synthesis and application of nanosize materials.
Nanostructures based on natural polysaccharides have been of particular interest in light of their
good biocompatibility, biodegradability, reduced toxic side effects and improved therapeutic
effects. Another advantage of using polysaccharides is that these molecules contain reactive
groups which can be used to introduce different chemical ligands. Marine polysaccharides such
as alginates, chitosans, carrageenans and fucoidans are examples of polymers that have been
studied for nanoparticles syntheses and that have shown promising results in several
applications. Alginate and chitosan nanostructures have been recently proposed as a system for
sustained release of several drugs, construction of biosensors such as immunosensor for the
detection of Escherichia coli in food specimens. Chitosan nanoparticles have been used to
remove heavy metals in a water treatment process; chitosan/alginate nanocomposites was
reported to be a good candidate for oral delivery of bioactive peptides; carrageenan and fucan
nanoparticles showed great spread ability and water-holding capability, a relevant method to
modulate interactions of the nanoparticles with several cells including tumor cells. The present
paper will reviewed recent progress in marine polysaccharide nanotechnology that presents itself
as the vanguard of the development of intelligent systems for biotechnology.
* E-mail address: [email protected]
60
1. INTRODUCTION
The term nanotechnology was proposed by Norio Tanaguchio (University of Tokyo) in 1974
when he referred to the ability of certain materials to form stable structures at nanometer scales.
Currently the term nanotechnology can be defined as the design and manufacture of materials,
device and systems with control at nanometer dimensions. But other more elaborate definitions can
be found. However, one should always bear in mind that the essence of nanotechnology is the size
control at the nanometer scale.
Nanotechnology represents a milestone in the sciences because of the possibility of producing
materials with nanometer-scale physical, chemical and biological exceptional. In this context
appears nanomedicine, which is the application of nanotechnology to medicine. This is a relatively
new term that has emerged in the last decades of the last century. This term covers procedures for
diagnosis, prevention and treatment of disorders and sequels, as well as preserving and improving
human health using molecular tools and molecular knowledge of the human body. Currently
nanomedicine exploits carefully structured nanoparticles, such as dendrimers, carbon fullerenes
(buckyballs), magnetic nanoprobes and nanoshells to reach specific organ and tissue. These
nanoparticles can serve both for diagnosis and for specific therapies. It is believed that with the
development of nanotechnology in the coming years, more complex nanodevices will be produced to
have a broader use of nanoparticles in medicine [1].
There are several classes of chemical compounds which may be used in applications
nanomedicine, one of which is represented by polysaccharides. Polysaccharides are natural polymers
consisting of one or different kinds of monosaccharides. It constitutes a very diverse and highly
versatile class of materials that have the most varied possible applications [2]. The polysaccharides
can be found in several sources, present in almost all taxa (Table 1).
Table 1. Usual sources of some polysaccharides
Organism Polysaccharide Source
Seaweeds
Alginate/Fucans/Fu
coidans Brown algae
Agarans/Carrageen
ans/ Agar-Agar/
Agarose
Red algae
Animals
Hyaluronic Acid Bovine vitreous humor,
gallinaceous crest
Heparin Bovine lung and porcine
intestines
Quitin/Chitosan Carapaces of crustaceous
Fungi Glucans Pleurotus ostreatus, Agaricus
blazei
Bacterias
Xantan
Dextran
Gelan
Xanthomonas ssp
Leuconostoc spp
Sphingmonas elodea
Plants
Cellulose Eucalyptus, pine trees and
other plants
Xilan Several sources
Starch Corn, wheat, potato and
portions of other plants
Inuline Several sources
Guar gum Cyamopsis tetragonolobus
and Cyamopsis Psoraloides
Gum karaya Sterculia urens, S. tomentosa
and other species of Sterculia
61
Gum arabic Acacia senegal and other
species of Acacias
Nanostructured systems have an important role in future therapies because they are widely
described in the literature as systems which cross the barrier-acting gastrointestinal epithelial
absorption. In addition, nanoparticles are able to control and direct the release of bioactive
substances. In nanomedicine, a drug (or a synthetic biopolymer) may be dissolved, incorporated,
encapsulated and adsorbed or bound to nanoparticles in order to improve their pharmacokinetic
profile, increase treatment efficiency, reduce adverse effects of preferential accumulation at specific
sites, so that there is low concentrations in healthy tissues; increase the chemical and conformational
stability of a variety of therapeutic agents such as small molecules, peptides and oligonucleotides.
The history of polysaccharides in nanotechnology begins with cellulose. Subsequently,
nanoparticles of other polysaccharides, such as hyaluronic acid, inulin, dextran and were being
described. Based on this, the synthesis of polysaccharide nanoparticles have been the focus of
relevant studies, compared to several other distribution systems, they show exceptional stability, and
respond very efficiently to biological systems in addition to providing low cytotoxicity and an
inexpensive obtaining process, thus opening a range of opportunities for the development of a
variety of biomedical applications, like as cell imaging, drug loading (Figure 1). Regarding the
polysaccharides of marine origin, the study of their use in nanotechnology has increased in recent
years and gained prominence in the scenario of cutting-edge research for numerous areas of science
[3]. Therefore, efforts were concentrated in this chapter in order to review information about the use
of polysaccharides of marine origin: carrageenan, fucoidans, quiosanas and alginic acids as raw
material for the production of different types of nanoparticles and their applicability.
Figure 1. The schematic represents the versatility of nanoparticles polysaccharides. Different regions of the
nanoparticles could act distinct roles in biomedical applications.
2. CARRAGEENAN
2.1. Chemical Structure
Carrageenan are a group of natural polysaccharides extracted from red seaweed (Rhodophyceae)
having particularity of forming colloids and gels in aqueous media at very low concentrations. These
gels are thermoreversible transparent and exhibit a wide range of textures, ranging from very elastic
and cohesive gel to firm and brittle, depending on the properties of the carrageenan used.
62
The carrageenan are known since the eighteenth century, but only in the fourteenth century they
started being extracted from red seaweed Chondrus crispus and used as an emulsifier and gelling
agent in homemade food by Irish population of the city of "Carrageen", where the name carrageenan
originated. Today carrageenan is one of the most used hydrocolloid worldwide, already surpassing
other polysaccharides such as alginates, agar.
The production of carrageenan was originally dependent on the natural deposits, especially C.
crispus (popularly known as "Irish Moss"), with a limited resource base. Since the early 70s of the
twentieth century, the industry has been rapidly expanding the availability and possibility of
cultivation of algae producing carrageenan in other hot water countries, with low labor cost.
Currently, most of the algae used for the production of carrageenan is from cultivations, although
there is still some demand for "Irish Moss," originating from natural deposits in Europe and Canada
and certain other types of algae yet uncultivated of South America. Current carrageenan production
is estimated at $ 240 million annually. Global demand has been growing around 5% annually over
the past 30 years, with prices ranging from $ 10 to $ 30 a pound, depending on their specifications
and quality. Europe has the largest market for carrageenan (55%). However, growth of its use is
strongly expected in regions such as Central America and South and Southeast Asia, where
consumption of carrageenan is expected to increase by 50% in the coming years.
The primary structure of carrageenan is a linear structure that is based on alternating copolymers
connections with β-1,3-D-galactose and α-1,4-D-galactose, with variable degrees of sulfation. The
units are joined together by alternating glycosidic bonds α-1, 3 and β-1, 4 which form the
disaccharide repeating unit of carrageenan. In some carrageenan a α-galactose can be dehydrated
giving rise to 3,6-Anhydro-α-D-galactose, there is a possibility of monosaccharides hydroxyls to be
substituted by methyl or pyruvate. These structural variables lead to the theoretical possibility of 42
different carrageenans. However, only 15 structures have been identified to date (Table 2).
Table 2. Disaccharide repeating structures of carrageenan a
Family Greek
symbol
1,3-linked 1,4-linked
Kappa Kappa (κ ) β-D-galactose 4-
sulfate
3,6-anhydro-α-D-galactose
Iota (ι) β-D-galactose 4-
sulfate
3,6-anhydro-α-D-galactose
2-sulfate
Mu (μ) β-D-galactose 4-
sulfate
α -D-galactose 6-sulfate
Nu (ν) β-D-galactose 4-
sulfate
α -D-galactose 2,6-di-
sulfate
Omicron
(ο)
β-D-galactose 4-
sulfate
α -D-galactose 2-sulfate
Beta Beta (β) β-D-galactose 3,6-anhydro-α-D-galactose
Gamma (γ) β-D-galactose α -D-galactose 6-sulfate
Omega (ω) β-D-galactose 6-
sulfate
3,6-anhydro-α-D-galactose
Psi (ψ) β-D-galactose 6-
sulfate
α -D-galactose 6-sulfate
Lambda Delta (δ) β-D-galactose α -D-galactose 2,6-di-
sulfate
Alfa (α) β-D-galactose 3,6-anhydro-α-D-galactose
2-sulfate
Lambda (λ) β-D-galactose 2- α -D-galactose 2,6-di-
63
sulfate sulfate
Theta (θ) β-D-galactose 2-
sulfate
3,6-anhydro-α-D-galactose
2-sulfate
Xi (ξ) β-D-galactose 2-
sulfate
α -D-galactose 2-sulfate
Pi (π) β-D-galactose P,2-
sulfate
α -D-galactose 2-sulfate
aAdapted from [4], P: pyruvate acetal
Due to their half-ester sulfate moieties, the carrageenan are anionic polymers strongly. The most
common types of carrageenan are traditionally called kappa (κ), iota (ι) and lambda (λ) [5]. They
differ only in the number of sulfate groups per disaccharide: κ have one, have two γ and λ is three
(Figure 2). The κ-carrageenan has a negative charge per disaccharide unit and presents the best
gelling properties among the three most common types of carrageenan [6].
Figure 2. Structural units of the three main types of carrageenans: (A) κ-carrageenan, (B) ι-carrageenan and (C) λ-
carrageenan. The disaccharides of κ-, ι- and λ-carrageenan are depicted, showing the β-1→4 and the α-1→3 bonds.
2.2. Nanoparticles of Carrageenan
In many types of nanoparticles, the carrageenan are not used as the main raw material, but as
polymers which cover the surface of the nanoparticle by giving it different characteristics from
inside, as well as protecting them from the external environment.
Colloidal nanocapsules (NC) have shown great potential as releasing peptides, lipophilic drugs
and vaccines in various types of mucus. NCs are composed of central oil by a surfactant and a
hydrophilic compound covering the entire surface of the NCs. During NCs formation or in a
subsequent incubation step, a water soluble polymer bearing an opposite electrical charge to that of
the surfactant can be incorporated by the ionic coat interaction, effectively yielding a stable colloidal
core-shell nanocapsule structure. One of the most promising hydrophilic polymers that have been
used in the production of NCs is carrageenan, mainly κ-carrageenan. These polysaccharides in NCs
are used at very low concentrations and the concentration that is used influences the size of the NCs.
64
Moreover, the negative charges of carrageenan stabilize the positive charge of the surfactant used
which can reduce cytotoxicity of surfactant related to its property to create pores in cells.
Metallic nanoparticles have a wide range of applications, but its low stability and size
distribution and heterogeneous form in aqueous represent a major problem for their use. These
problems can be minimized by coating the nanoparticles with polysaccharides. Daniel-da-Silva and
coworkers (2007) have synthesized supramagnetic nanoparticles of Fe3O4 in the presence of three
different types of carrageenan through a co-precipitation method [7]. Besides avoiding spontaneous
agglomeration of the nanoparticles, the carrageenan promoted the formation of smaller particles.
These authors also showed that the size and shape of the nanoparticles were influenced by the type
of carrageenan (kappa, iota, or lambda) as the concentration of polysaccharide used. Those particles
formed with iota-carrageenan were the most stable. Potential applications of such nanoparticles
include separation and cell labeling, contrast enhancement in magnetic resonance imaging (MRI),
controlled delivery of drugs and treatment of cancers by hyperthermia.
During the production of this chapter there were no examples found in the literature of
nanoparticles synthesized exclusively from carrageenan. However, nanoparticles synthesized from
carrageenan associations with other polysaccharides have been found, which gives the particle very
interesting characteristics obtained for drug delivery. Like for example, carrageenan and chitosan
nanoparticles. These nanoparticles are produced under hydrophilic conditions by a very mild process
of ionic interaction between the amino groups of chitosan positively charged and negatively charged
sulfate groups of the carrageenan. This procedure avoids the use of organic solvents and other
aggressive conditions which may be detrimental to the integrity of the drug to be released. Studies
show that the carrageenan-chitosan nanoparticle have an associated load capacity ranging from 4%
to 17% and excellent capacity to provide a controlled release of drug over a prolonged period of 3
weeks. In addition, these nanoparticles have shown no cytotoxic behavior in biological assays in
vitro when using L929 fibroblasts, which is critical of the biocompatibility of these carriers. The
development of carrageenan-chitosan nanoparticles has shown promising properties for use not only
as releasing agents, but also in other fields, such as tissue engineering and regenerative medicine [9].
3. FUCOIDAN
3.1. Chemical Structure
Fucoidan are heteropolysaccharides having in its constitution sulfated L-fucose therefore can
also be called heterofucan. Xylose, galactose, glucose, mannose, glucuronic acid are the other
monosaccharides that are commonly found in the constitution of fucoidan addition of fucose. There
are still some fucoidan showing fucose residues substituted by acetyls groups.
The first description of the purification of a fucoidan date of year 1913 and refers to acidic
polysaccharides extracted from brown seaweed Laminaria digitata, Fucus vesiculosus and
Ascophyllum nodosum. However, only in 1931 it was demonstrated the presence of sulfate groups in
fucose monomers. The understanding of these chemical structural compounds only intensified in the
50’s of last century.
Literature data show that fucoidan are structurally very complex molecules. They can have
different types and amounts of monosaccharides which may be interconnected by different types of
glycosidic bonds, although the connections 1-3 and 1-4 being the most common. Furthermore, most
of them are branched. This structural complexity is compounded by the fact that the amount and
location of sulfate substitution is different in each molecule, and may also occur in other
monosaccharides in addition to fucose. In some cases, there is the possibility that some of fucose
residues are acetylated.
The fucoidan are only synthesized by brown algae. However, because of structural complexity
as described in the previous paragraph, each algae synthesizes one or more type of fucoidan, which
is structurally different from that synthesized by any other brown algae, fact that causes the variety
65
of fucoidan (Table 3) found in nature to be high in comparison with other molecules such as
proteins, despite fucoidan are only synthesized by brown algae.
Table 3. Structural properties of some different fucoidan sources
This structural complexity partly justifies the diversity of biological activities and
pharmacological properties attributed to fucoidan such as antitumor, antiviral, immunomodulatory,
anticoagulant, anti-inflammatory, and antioxidant, [10, 22-25].
3.2. Fucoidans Nanoparticles and Its Application in Medicine
There is an increasing need to develop a reliable methodology and "Eco" for synthesizing metal
nanoparticles which can be used for many applications. Natural compounds such as fucoidans are
one means that can be used for this purpose and various metal ions can be used, such as iron, silver
and gold, among others.
66
Soisuwan et al (2010) using fucoidans from Cladosiphon okamuranus and Kjellamaniella
crassifolia were able to synthesize first gold nanoparticles coated with fucoidan [26]. The data
showed that the particles had an average size of 10 nm and that linear fucoidan C.okamuranus
nanoparticles were produced with less dispersion in size than the fucoidan K. crassifolia. In that
same year silver nanoparticles coated with fucoidan were also synthesized for the first time. In this
case the authors took advantage of the reductive capacity of fucoidan from Fucus vesiculosus and
managed to produce nanoparticles in an "environmentally friendly" way. The data show that the
particle size and its distribution were dependent on the concentration of fucoidan and silver used
during the synthesis process [27]. These nanoparticles are being tested as antimicrobials and as
components in sensors.
The fucoidan, as with the carrageenan are also used in the synthesis of nanoparticles associated
with other biopolymers or synthetic polymers. Nanoparticles formed by the combination of chitosan
(positive charges) and fucoidan (negative charge) from F. vesiculosus seaweed were prepared in
order to be resistant to the gastrointestinal environment. Currently, the oral route is considered the
most convenient and comfortable when administering medications to patients. However, the
significantly low pH gastric environment of the stomach causes deterioration of many drugs prior to
absorption. Therefore, conveyor systems resistant to gastrointestinal environment have been studied.
The data show that the best nanoparticles were obtained with the 1:1 ratio of fucoidan and chitosan.
This particle has been able to incorporate curcumin (an antitumor drug) and release it in greater
quantity only at pH above 6.0 [28].
Fucoidans-based nanoparticles have also been synthesized. Lira and colleagues (2011) have
synthesized nanoparticles with fucoidan extracted from seaweed Sargassum cymosum. These
particles showed low cytotoxicity against J774 macrophage and 3T3 fibroblasts. Confocal
microscopy studies showed that these nanoparticles are internalized by cells. The data also showed
that the interactions of nanoparticles with macrophages can be modulated by the introduction of
fucoidan in the center of the nanoparticles and future studies elucidate the mechanisms of uptake and
intracellular pathways of its trajectory [29].
Nanoparticles were synthesized with a fucoidan from the Spatoglossum schroederi seaweed.
The synthesized nanogels of about 123 nm showed high stability in size and conformation for 70
days. These particles inhibited the proliferation of human tumor cells in liver, kidney and bone tissue
being able to promote the activation of caspases and affect the distribution of these cells in different
cell cycle phases [30].
4. CHITOSANS
4.1. Chemical Structure
Chitosan (QS) is a linear polysaccharide derived from chitin, compound found in the
exoskeleton of some insects, fungi and marine invertebrates, such as shrimp and crab. The chemical
structure of chitosan molecule is very similar to cellulose and chitin. Chitosan is a linear random
polymer structure composed of backbone of β(1-4)-2 acetoamida-2-deoxy-D-glucopyranose and 2-
amino-2-deoxy-D-glucopyranose. The amino groups are protonated and this polysaccharide showed
positively charged at physiological fluids. The process of deacetylation of chitin to chitosan
formation is not complete and the degree of deacetylation of chitin, a polymer defines or chitosan,
the amount of acetoaminated groups in the chitosan is extremely low (degree of acetylation of less
than 0.35).
Chitin was discovered at the beginning of the 19th
century by French scientist Henri Braconnot.
But the QS was only discovered in the 80’s of the 20th
century. Commercially chitosan is obtained
from the deacetylation of chitin extracted from crab and shrimp shells.
67
4.2. Chitosan nanoparticles (NP-QS)
Many of the polymers used today in many areas, are synthetic materials and their
biocompatibility and biodegradability are very limited. Moreover, the QS is one of the most
abundant natural polysaccharide and well known for its biodegradability and non-toxic properties.
Also, the QS has unique characteristics as a dependent pH behavior, dependent conformational
variability of the environment in which is found, mucus adherence and ease of overcoming the
epithelial junctions [31]. These characteristics makes the use of QS already performed in various
fields like biomaterials, pharmaceuticals and cosmetics, sequestration of metal ions, agriculture,
foodstuff and treatments (clarification, flocculation, etc., due to their effective interaction with other
polyelectrolytes) [32]. However, due to the property of QS, the biotechnological potential can be
much broader and its applications can be extended to other fields of human activity. A clear example
of the search for new applications for the QS is a quantity of scientific articles that focused primarily
nanotechnology and QS. Currently, there are approximately 10,000 articles related to QS
nanoparticles, accumulating more than 119,000 citations; these data show the great interest by the
scientific community in developing QS nanoparticles. In 2011, more than 1400 articles have been
published relating to nanotechnology chitosans (Figure 3) [31].
Figure 3. Scientific impact of NP-QS. The graphs represent annual perspective of (a) numbers of publications by year
related to the topic, and (b) shows the number of citations referring to articles published related to the topic. Boolean
search defined: Topic = (chitosan) AND Topic =(nanoparticle* OR nanosphere* OR complex* OR nanocarrier*)
AND Topic = (chitosan* OR nanoparticles* OR nanomedicine*); Languages = (ENGLISH); Timespan = All Years.
Databases = SCI-EXPANDED, SSCI, NCBI, SCIENCEDIRECT. Retrieved on October, 18th, 2012.
Among this substantial amount of scientific articles relating nanotechnology and QS, there is a
great emphasis on those who are targeted for nanomedicine, especially for applications related to
transporting molecules as drugs, proteins, hormones, nucleic acids and hydrophobic molecules,
besides its use in tissue engineering and diagnosis of disease assays. However, you can find articles
evaluating the potential of QS in less usual areas for polysaccharides such as in the production of
biosensors.
The drug delivery systems (DDS - Drug Delivery Systems) are being intensely investigated in
recent years due to its great potential to enhance the therapeutic index of small molecules. Among
these systems major advances have been achieved in the area: i-Vaccination; ii- Transmucosal
Peptide/ Protein Delivery; iii-Controlled Release Drug and iv- Gene Therapy.
Nanocarriers drug to be used as DDS has as an important feature the ability to form a porous
structure that allows the input and specially the output of drugs, and this characteristic is easily
obtained with the chitosan nanoparticles (NP-QS) including when using simple techniques, low cost,
as boiling internal process [33]. Moreover, NP-QS has other important features that drive their DDS
studies as they exhibit biodegradability, low cytotoxicity, antibacterial activity, permit the addition
covalently easily, other molecules its structure and mucus adherence. These NP-QS properties
provided the development of numerous oral, ocular, nasal, vaginal, intra-vesicular systems for the
drift, targeting and controlled release of drugs and other molecules in the body of animals, including
humans [34, 35].
68
For the production of NP-QS many techniques can be used, as nanocomplexity, formation of
ianotrophic icing mixtures, layer-layer capsulation, water-oil emulsification, membrane
emulsification, compression, casting, adsorption processes. This QS plasticity may be posted on
different techniques for producing nanoparticles partly explains the large number of articles on NP-
QS related carriers of drugs. Another important factor is that QS can be modified chemically to
improve its solubility in certain solvents and encourage interaction with specific drugs or other
molecules such as nucleic acids and proteins, enhancing the development of NPs appropriate to the
compound to be loaded [36]. Furthermore, in the development of NP-QS, QS can be combined with
a variety of other materials such as alginates, fucoidan, carrageenan, hidroxihapatitas, hialunonic
acid, calcium phosphate, poly (ɛ-caprolactone (PCL), peptides and factors growth, so that it can be
modular action of polymers that comprise the nanoparticle and thus uses them in release systems.
As early as the mid 90’s of the last century it was possible to identify several data showing the
use of QS NP-carriers of drugs and other molecules such as, for example, a system combining
nanocapsules polycaprolactone as carriers and advantages of mucus adhesive chitosan and cationic
PLL (poly-L-lysine) was prepared with the aim of increasing viability of ocular drugs. Although
both the PLL as chitosan show a similarity in positively charged surface, only chitosan nanocapsules
covered with increased ocular penetration of indomethacin in relation with the not covered
nanocapsules [37].
During the last two decades studies on QS nanoparticles were intensified which greatly
increased the knowledge about the synthesis methodologies and their mechanisms of action. At the
beginning of the century, Mitra and colleagues proposed the use of chitosan nanoparticles to
improve the therapeutic efficacy of doxorubicin (DXR), a widely used drug in the treatment of solid
tumors, but has undesirable side effects such as cardiotoxicity. DXR was conjugated to dextran (to
facilitate the incorporation of the drug nanoparticles and QS incorporated into the chitosan
nanoparticles (100 ± 10 nm in diameter) and were then evaluated in vivo in Balb c mice with tumor
[38]. Use of chitosan nanoparticles as carriers increased the survival of the animals compared to the
free drug [38]. In the following years, some work showed the development of knowledge in the
production methodology of NP-QS and incorporation of different molecules to the nanoparticles
produced. As the work of Ma and colleagues who demonstrated the importance of pH on the
incorporation efficiency of NP-QS insulin, indicating that the next at pH 6 the amount of insulin
incorporated fold [39]. After, Sarmento and colleagues in an assay using rodents, showed that blood
glucose levels were significantly diminished when nanoparticles of chitosan/alginate load with
insulin were orally administered [40].
Banerjee and colleagues in 2002 report the importance of amine groups in the size of chitosan
nanoparticles. The QS-NP had a size of ± 30 nm in diameter when ± 10% of amino groups were
linked (using glutaraldehyde as an agent for the cross-linked) and upper diameter of 110 nm where
virtually all groups were linked [41]. The loads on positive QS proved sums to the properties
displayed by NP-QS, as observed by El-Shabouri (2002), who found that the incorporation of
cyclosporin A (Cly-A) in NP-QS proved very effective when compared to glycocholate
nanoparticles (SGC); the author has shown that positively charged nanoparticles improved the
viability of oral Cly-A by approximately 73%, since the NP-SGC decreased by about 36% [42].
More recently, Sonaje and colleagues have proposed that NP-QS containing drugs can adhere and
passed through intestinal mucus; the infiltrated NP-QS become unstable and disintegrate near the
epithelial cell surface due the microenvironment pH and thus release the loaded drug [43]. Thus, the
released drug could then enter the systemic circulation due to the QS-mediated TJ opening (Figure
4), which could explain the effect of NP-QS to improve the drug oral viability.
69
Figure 4. NP-QS intestinal absorption and drugs release. Nanoparticles are capable of passing through the
mucosal barrier produced by epithelial cells and release the drug in the intracellular space because of their pH-
induced structural change.
Other studies suggested the use of NP-QS for topical use in genetic immunization. To propose
this, Cui and Mumper (2001) incorporated a plasmid (pDNA) containing luciferase and specific IgG
antigen in NP-QS and subsequently administered this nanoparticle topically. Data showed a
significant luciferase expression in the skin epidermis of mice that underwent topical application of
NP-QS loaded with DNA, indicating a significant efficiency in the introduction of pDNA in the
nucleus of epithelial cells when carried by NP-QS. Moreover, there is a significant serum IgG titer to
the antigen specific for β-galactosidase expression after 28 days of application of NP-QS/pDNA
[44].
One of the qualities of a system for controlled release of drugs is the possibility of continuous
analysis of drug delivery to their specific location. Using the principle of electron transfer and
energy transfer fluorescent resonance (FRET), new models of chitosan nanoparticles have been
developed to monitor the release of drugs. By associating within the nanoparticle drug interacts with
the particle preventing the release of fluorescent light freedom to be determined, the drug in the
nanoparticle site in the body returns to the initial state and emits fluorescent light indicating the
release of the drug [45].
Magnetic/luminescent QS Nanogels were synthesized by direct icing of chitosan, fluorescent
particles (CdTe quantum dots) and iron oxide supermagnetic nanogels inside and subsequently
loaded with insulin. The incorporation of these nanoparticles by the cells seems to be mediated by
insulin receptors and the high incorporation efficiency of these chitosan nanogels directed to the
liver cells and a high viability of these cells after using nanogel (above 80%) of NP-QS is a potential
tool for targeted drug delivery, cell imaging research and delivery of targeted diet supplements [46].
The use of chitosan nanoparticles as carriers of small molecules systems makes them a potential
tool for immunization against various microorganisms. Intranasal administration of nanoparticles
(NPs) of poly gama-glutamate/chitosan recombinants loaded with antigens of influenza virus
hemagglutinin (rHA) or inactivated virus is able to reach the mucous membrane, such as mucosal
adjuvant induces a high degree of protective immunity mucus of the respiratory tract. Intranasal
administration of rHA or inactivated virus antigen loaded NPs were able to protect mice when a
lethal dose of highly pathogenic influenza (H5N1 virus) was injected in animals [47]. Other studies
have demonstrated that NP-QS alone already present as agents that can combat micro-organisms, as
70
demonstrated by Shiet and coworkers (2006) [48]. These authors showed that NP-QS had
bactericidal activity against Staphylococcus aureus and Staphylococcus epidermidis.
Chitosan nanoparticles can also act as biosensors aiding in the diagnosis of various diseases.
Carbon nanotubes were used as biosensors; its multiple walls modified by adding magnetic
nanoparticles of chitosan and improved the accuracy and reproducibility sensitivity quantification of
human serum albumin from hospital samples [49]. NP-QS can act as diagnostic imaging when
directed specifically to certain targets in the body, such as tumor cells. A new magnetic nanoparticle
formed from the "layer-layer" technique (shown) the combined use of iron oxide nanoparticles (core
of the nanoparticle), followed with layers of chitosan, fluorescent particles (CdTe quantum dots), a
new layer chitosan, folate layer (targeting cellular receptors for folate) and adiamicine (an
antitumour), that nanoparticles were effective in releasing the drug targeting to tumor cells, and are
easily detected because of the localized presence of fluorescent molecules [46].
Besides the use in biomedical systems, chitosan nanoparticles can be developed and adapted for
use as environmental clean-up. NP-SQ has been developed for the removal of heavy metals and dyes
from aqueous environments. Metals such as mercury, copper and zinc were efficiently removed (to
98% removal of mercury) from aqueous solutions using a magnetic chitosan resin [50]. In a study
using magnetic nanoparticles coated with chitosan-β-cyclodextrin (CDC) it was possible to remove
approximately 90% of methyl blue dye aqueous solution. The CDC grafted onto the Fe3O4
nanoparticles contributes to an improvement in adsorption capacity due to the presence of several
hydroxyl groups, carboxyl groups, amino groups and formation of inclusion complexes to absorb the
dye. Adsorption shown to be dependent on pH and temperature and with magnetic chitosan
nanoparticle was stable and easily recovered and reused [51].
5. ALGINATES
5.1. Chemical Structure
Alginate is a term used to describe salts of alginic acid, but is commonly used for all derivatives
of alginic acid and including itself. In brown algae, alginates promote the required flexibility for the
growth conditions of the algae in the marine environment. Naturally, they form salts with gelatinous
characteristics when combined with the minerals in sea water.
Alginates (ALG) are unbranched polysaccharides consisting of four links between 14 β-D-
mannuronic acid (M) and its C-5 epimer α-L-glucuronic acid (G); these monomers may be present in
homopolymeric blocks of consecutive M (M blocks) or G (G blocks) or alternating M and G
monomers (MG blocks). This natural polymer is an important component of brown algae cell wall,
and also an exopolysaccharide of bacteria such as Pseudomonas aeruginosa.
Alginates are the most abundant heteropolysaccharides of brown algae comes to constitute about
40-60% of the dry weight of certain species of seaweed. Most are commercially extracted from the
brown algae Macrocystis pyrifera (Pacific Coast in America) and Ascophyllum nodosum (Europe).
But other species also deserve mention, as Laminaria digitata, Laminaria hyperborea, and several
species of Sargassum.
In 1883, Dr. E. C. C. Standford, Scottish scientist, was the first to isolate and use the name
alginic acid, since then, many researchers have been devoted to the study of these molecules with
multifunctional and comprehensive biotechnological potential. The industrial exploitation of algal
alginates is directly linked to their properties: ability to form gel, change the viscosity of the
solutions; films form (calcium or sodium salts) or fiber (calcium salt)
ALG have been found in many types of applications in the biomedical and engineering due to its
favorable characteristics such as biocompatibility and easy icing using simple techniques. Alginates
are particularly attractive for use as wound healing, drug delivery systems and other small
molecules, and tissue engineering, since the gels formed from such polymers hostage a similar
structure to the extracellular matrix in tissues and also may be manipulated to play many important
71
roles. Among the pharmacological properties of alginates, it stands out its anti-ulcer because the
alginate forms a protective layer of the gastric wall preventing the action of stomach acids in their
walls. There is also its application as a material which coats capsules and controlled release of drugs
in the treatment of skin ulcers, acting as a barrier between the organ and the immune system, among
others.
5.2. Nanoparticles of Alginates
The proportion of MG blocks in the alginate molecule appears to influence the properties of the
polymer for forming magnetic nanoparticles of iron. Alginate rich in fatty guluronic ratio (M / L <1),
such as those found in species Sargassum retains large amount of iron in the loop formed, and thus
form larger amount of nanoparticles of iron oxide compared to other alginates [52].
Beyond the easy synthesis methodology nanoparticles ALG, the polymers can also form meshes
micrometric that facilitate the production of other types of nanoparticles, as demonstrated by Wang
and coworkers, in 2012 [56]. They have proposed a new method for the production of barium sulfate
nanoparticles (NP-BaSO4) by using microgels Ba-ALG, the mesh alginate served as a template
lightweight and porous prevented precipitation and aggregation of nanoparticles BaSO4. Due to the
NP-opaque characteristics BaSO4, these structures can act as radiopaque agents in the area of
diagnostic imaging [53].
Great efforts have been directed to the application of nanoparticles with carrier agents, following
the trend in the area of controlled release of drugs and other molecules knowledge on NP-ALG as
carriers, which has increased significantly in the last decade. In 2002, González-Rodríguez and
colleagues used a system of nanoparticles of alginate/chitosan for controlled release of diclofenac
sodium. The nanoparticles were synthesized through an icing method using calcium ion (Ca2 +) and
Aluminum (Al3 +) and loaded with sodium diclofenac. It was demonstrated that drug release was
dependent of pH where NPs at pH acid drug release did not occur; moreover, around pH 6.4-7.2 was
completely released [54]. In the following year, and Robinson published a study which showed the
importance of the proportions of components (such as alginate, PLL , chitosan and calcium chloride)
in the synthesis of nanoparticles alginate, being such concentrations critical for the formation of
nanospheres or microspheres. A mass ratio of less than 0.2 between calcium chloride and alginate
was essential for the formation of nanospheres as well as adding another cathonic product as
chitosan and PLL [55].
Over the last decade more data were generated and different methodologies were used to
develop ALG nanoparticles. Recent works have shown that the NP-ALG may be used as efficient
delivery systems and release of drugs and other small molecules. NP-ALG (ALG-PDEA) (alginic
acid / poly [2 - (diethylamino) ethyl methacrylate]) were synthesized and loaded with doxorubicin
(DOX) and evaluated for antitumor activity in vitro and in vivo through cell and animal culture
model. The results of speed and amount of in vitro release of DOX from ALG-PDEA increase when
the pH of the medium decreases. Furthermore, in vivo results showed that NPs were efficiently
incorporated into tumor cells, benefiting from the increased permeability of solid tumors and
retaining (EPR effect). In addition, NPs loaded significantly suppress the tumor as compared to free
DOX and bio-distribution studies showed that DOX was increased at the tumor site and longer blood
circulation in animals, and still lesser concentration of drug in heart and lung, thereby reducing the
inherent toxicity of DOX [59]. Other data from the same research group using a new method of NP-
ALG synthesized using alginate (negatively charged) and 2,2 - (ethylenedioxy) bis (ethylamine)
(positively charged and then the addition interlinking calcium agent (cross-link) and then loaded
with DOX. These 100 nm nanoparticles showed zeta potential of-30mV, were to be incorporated
within the tumor cells, increased drug bio-distribution and accumulation at the tumor site. Moreover,
the data showed interlinking agent (Ca2 +) in stabilization of NPs in an environment at physiological
pH [56].
72
The ALG can also be associated with other polysaccharides of marine origin for the preparation
of nanoparticles. NPs sodium alginate / chitosan were loaded with 5-fluorouracil (5-FU) and
evaluated as drug delivery systems for ophthalmic use. In this work it became evident that the molar
relationship between chitosan and alginate are fundamental to the size of the nanoparticle and the
drug encapsulation efficiency of the NP. The in vivo results showed an increase in the effective
concentration of 5-FU in the aqueous humor as compared with a solution of 5-FU revealing an
effective topical application to ocular NP-ALG combined with chitosan [57].
With the increased number of work and knowledge of nanoparticles of alginate, new
applications have been proposed for the NP-ALG. Silver nanoparticles / alginate (AgNP/ALG) were
synthesized and used for drinking water disinfection. AgNP/ALG were synthesized using three
different couplers as filter material packed columns for simultaneous filtration and disinfection as an
alternative for the treatment of drinking water. When compared to silver particles, the AgNP/ALG
able to eliminate satisfactorily E. Coli and produce a smaller amount of silver deposit, being thereby
more effective and less toxic than the silver-only [58].
CONCLUSION
Marine polysaccharide nanomedicine technology has reached considerable maturity in the last
20 years. There is now an extensive body of intellectual property related to polysaccharides based
nanomedicines, and compelling evidence for the potential of marine polysaccharides nanoparticules
for many challenging drug delivery and tissue engineering applications. Currently, there is a broad
wisdom network about nanoparticles from marine polysaccharide and compelling evidence for the
potential nanocarriers to drug delivery, gene silencing or immunomodulatory peptides release and
even other applications, like image diagnostic. The structural diversity of polysaccharides allows
chemical modifications and the nanoproducts physicochemical features can be shaped to connect
with different cellular targets and also improve the bio-compatibly which improves use efficiency of
a particular drug and also dramatically reduces the side effects. Despite promising in vitro results
have been achieved over the last ten years, and optimistic data has been developed in human trials
exhaustive in vivo researches are necessary to address challenges in drug human administration and
then could be used in wide scale for therapeutic or even preventive measures. However, many
authors are confident that in short time based-marine polysaccharides nanoproducts will enter the
market.
73
REFERENCES
[1] Freitas Jr, R. A. (2005). Nanotechnology, nanomedicine and nanosurgery. International
Journal of Surgery, 3 (4), 243-246.
[2] Da Cunha, P. L. R.; De Paula, R. C. M. & Feitosa, J. P. A. (2009). Polissacarídeos da
biodiversidade brasileira: uma oportunidade de transformar conhecimento em valor
econômico. Quimica Nova, 32 (3), 649–660.
[3] Gil, M. H. & Ferreira, P. (2006). Polissacarídeos como biomateriais. Quimica, 100, 72-74.
[4] Lahaye, M. (2001). Developments on gelling algal galactans, their structure and physico-
chemistry. Journal of applied phycology, 13, 173–184.
[5] Santo, V. E.; Frias, A. M.; Carida, M.; Cancedda, R.; Gomes, M. E.; Mano, J. F. & Reis, R. L.
(2009). Carrageenan-based hydrogels for the controlled delivery of PDGF-BB in bone tissue
engineering applications. Biomacromolecules, 10 (6), 1392–1401.
[6] Coviello, T.; Matricardi, P.; Marianecci, C. & Alhaique F. (2007). Polysaccharide hydrogels
for modified release formulations. Journal of Controlled Release, 119 (1), 5–24.
[7] Daniel-da-Silva, A. L.; Trindade, T.; Goodfellow, B. J.; Costa, B. F. O.; Correia, R. N. & Gil,
A. M. (2007). In situ synthesis of magnetite nanoparticles in carrageenan gels.
Biomacromolecules, 8, 2350–2357.
[8] Grenha, A.; Gomes, M. E.; Rodrigues, M.; Santo, V. E.; Mano, J. F.; Neves, N. M.; Reis, R.
L. (2010). Development of new chitosan/carrageenan nanoparticles for drug delivery
applications. Journal Biomedical Material Research Part A, 92 (4), 1265-1272.
[9] Albuquerque, I. R. L.; Queiroz, K. C.; Alves, L. G.; Santos, E. A.; Leite, E. L. & Rocha, H. A.
(2004). Heterofucans from Dictyota menstrualis have anticoagulant activity. Brazilian Journal
of Medical and Biological Research, 37 (2), 167–171.
[10] Li, B.; Lu, F.; Wei, X. & Zhao, R. (2008). Fucoidan: structure and bioactivity. Molecules, 13
(8), 1671–1695.
[11] Leite E. L.; Medeiros, M. G. L.; Rocha, H. A. O.; Farias, G. G. M.; Silva, L. F.; Chavante, S.
F.; Abreu, L. R. D.; Dietrich, C. P. & Nader, H. B. (1998). Structure and pharmacological
activities of a sulfated xylofucoglucuronan from the alga Spatoglossum schröederi. Plant
Science, 132, 215–228.
[12] Rocha, H. A. O.; Moraes, F. A.; Trindade, E. S.; Franco, C. R. C.; Torquato, R. J.; Veiga, S.
S.; Valente, A. P.; Mourão, P. A.; Leite, E. L.; Nader, H. B. & Dietrich, C. P. (2005).
Structural and hemostatic activities of a sulfated galactofucan from the brown alga
Spatoglossum schröederi. An ideal antithrombotic agent? The Journal of Biological
Chemistry, 280 (50), 41278–41288.
[13] Adhikari, U.; Mateu, C. G.; Chattopadhyay, K.; Pujol, C. A.; Damonte, E. B. & Ray, B.
(2006). Structure and antiviral activity of sulfated fucans from Stoechospermum marginatum.
Phytochemistry, 67, 2474–2482.
[14] Maraias, M. F. & Joseleau, J. P. (2001). A fucoidan fraction from Ascophyllum nodosum.
Carbohydrate Research, 336 (2), 155–159.
[15] Bilan, M. I.; Zakharova, A. N.; Grachev, A. A.; Shashkov, A. S.; Nifant’ev, N. E. & Usov, A.
I. (2007). Polysaccharides of algae: 60. Fucoidan from the Pacific brown alga Analipus
japonicus (Harv.) Winne (Ectocarpales, Scytosiphonaceae). Bioorganicheskaia Khimiia, 33
(1), 44–53
[16] Bilan, M. I.; Grachev, A. A.; Shashkov, A. S.; Nifantiev, N. E. & Usov A. I. (2006). Structure
of a fucoidan from the brown seaweed Fucus serratus L. Carbohydrate research, 341 (2), 238–
245.
[17] Li, B.; Xin, J. W.; Sun, J. L. & Xu, S. Y. (2006). Structural investigation of a fucoidan
containing a fucose-free core from the brown seaweed Hizikia fusiforme. Carbohydrate
Research, 341 (9), 1135–1146.
74
[18] Chizhov, A. O.; Dell, A.; Morris, H. R.; Haslam, S. M.; McDowell, R. A.; Shashkov, A. S.;
Nifant'ev, N. E.; Khatuntseva, E. A. & Usov, A. I. (1999). A study of fucoidan from the brown
seaweed Chorda filum. Carbohydrate Research, 320 (1-2), 108–119
[19] Usov, A. I.; Smirnova, G. P.; Bilan, M. I. & Shashkov, A. S. (1998). Polysaccharides of algae:
53. Brown alga Laminaria saccharina (L.) Lam. as a source of fucoidan. Bioorganicheskaia
Khimiia, 24, 382–389.
[20] Lee, J. B.; Hayashi, K.; Hashimoto, M.; Nakano, T. & Hayashi, T. (2004). Novel antiviral
fucoidan from sporophyll of Undaria pinnatifida (mekabu). Chemical & Pharmaceutical
Bulletin, 52 (9), 1091–1094
[21] Kim, W. J.; Kim, H. G.; Oh, H. R.; Lee, K. B.; Lee, Y. K. & Park, Y. I. (2007). Purification
and anticoagulant activity of a fucoidan from Korean Undaria pinnatifida sporophyll. Algae,
22 (3), 247–252.
[22] Cumashi, A.; Ushakova, N. A.; Preobrazhenskaya, M. E.; D'Incecco, A.; Piccoli, A.; Totani,
L.; Tinari, N.; Morozevich, G. E.; Berman, A. E.; Bilan, M. I.; Usov, A. I.; Ustyuzhanina, N.
E.; Grachev, A. A.; Sanderson, C. J.; Kelly, M.; Rabinovich, G. A.; Iacobelli, S. & Nifantiev,
N. E. (2007). A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic,
and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology, 17
(5), 541–552.
[23] Tanaka, K.; Ito, M.; Kodama, M.; Tomita, M.; Kimura, S.; Hoyano, M.; Mitsuma, W.; Hirono.
S.; Hanawa. H. & Aizawa, Y. (2011). Sulfated polysaccharide fucoidan ameliorates
experimental autoimmune myocarditis in rats. Journal of cardiovascular pharmacology and
therapeutics, 16, 79–86.
[24] Costa, L. S.; Fidelis, G. P.; Telles, C. B.; Dantas-Santos, N.; Camara, R. B.; Cordeiro, S. L.;
Costa, M. S.; Almeida-Lima, J.; Melo-Silveira, R. F.; Oliveira, R. M.; Albuquerque, I. R.;
Andrade, G. P. & Rocha, H. A. (2011). Antioxidant and antiproliferative activities of
heterofucans from the seaweed Sargassum filipendula. Marine drugs, 9 (6), 952–966.
[25] Morya, V. K.; Kim, J. & Kim, E. K. (2012). Algal fucoidan: structural and size-dependent
bioactivities and their perspectives. Applied microbiology and biotechnology, 93 (1), 71–82.
[26] Soisuwan, S.; Warisnoicharoen, W.; Lirdprapamongkol, K. & Svasti, J. (2010). Eco-friendly
synthesis of fucoidan-stabilized gold nanoparticles. American journal of applied sciences, 7,
1038–1042.
[27] Leung, T. C.-Y.; Wong, C. K. & Xie, Y. (2010). Green synthesis of silver nanoparticles using
biopolymers, carboxymethylated-curdlan and fucoidan. Materials Chemistry and Physics, 121
(3), 402–405.
[28] Huang, Y.-C. & Lam, U.-I. (2011). Chitosan/Fucoidan pH Sensitive Nanoparticles for Oral
Delivery System. Journal of the Chinese Chemical Society, 58 (6), 779–785.
[29] Lira, M. C.; Santos-Magalhães, N. S.; Nicolas, V.; Marsaud, V.; Silva, M. P.; Ponchel, G.;
Vauthier, C. (2011). Cytotoxicity and cellular uptake of newly synthesized fucoidan-coated
nanoparticles. European journal of pharmaceutics and biopharmaceutics, 79 (1), 162–170.
[30] Dantas-Santos, N.; Almeida-Lima, J.; Vidal, A. A.; Gomes, D. L.; Oliveira, R. M.; Santos
Pedrosa, S.; Pereira, P.; Gama, F. M. & Oliveira Rocha, H. A. (2012). Antiproliferative
activity of fucan nanogel. Marine Drugs, 10 (9), 2002–2022.
[31] Casettari, L.; Vllasaliu, D.; Lam, J. K.; Soliman, M. & Illum, L. (2012). Biomedical
applications of amino acid-modified chitosans: A review. Biomaterials, 33 (30), 7565–7583.
[32] Laurienzo, P. (2010). Marine Polysaccharides in Pharmaceutical Applications: An Overview.
Marine drugs, 8 (9), 2435–2465.
[33] Chow, K. S. & Khor, E. (2000). Novel fabrication of open-pore chitin matrices.
Biomacromolecules, (1), 61–67.
[34] Suh, J. K. F. & Matthew, H. W. T. (2000). Application of chitosan-based polysaccharide
biomaterials in cartilage tissue engineering: A review. Biomaterials, 21, 2589–2598.
[35] Di Martino, A.; Sittinger, M. & Risbud, M. V. (2005). Chitosan: A versatile biopolymer for
orthopaedic tissue engineering. Biomaterials, 26, 5983–5990.
75
[36] Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A. & Benoit, J. P. (2008). Progress in
developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials,
29, 3477–3496.
[37] Calvo, P.; Vila-Jato, J. L. & Alonso, M. J. (1997). Evaluation of cationic polymer-coated
nanocapsules as ocular drug carriers. International Journal of Pharmaceutics, 153 (1), 41–50.
[38] Mitra, S.; Gaur, U.; Ghosh, P. C. & Maitra, A. N. (2001). Tumour targeted delivery of
encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles as carrier. Journal
Control Release, 74 (1-3), 317–323.
[39] Ma, Z.; Yeoh, H. H. & Lim, L. Y. (2002). Formulation pH modulates the interaction of insulin
with chitosan nanoparticles. Journal of pharmaceutical sciences, 91 (6), 1396-1404.
[40] Sarmento, B.; Ribeiro, A.; Veiga, F.; Sampaio, P.; Neufeld, R.; Ferreira, D. (2007).
Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharmaceutical research,
24 (12), 2198–2206.
[41] Banerjee, T.; Mitra, S.; Kumar, S. A; Kumar, S. R. & Maitra, A. (2002). Preparation,
characterization and biodistribution of ultrafine chitosan nanoparticles. International journal of
pharmaceutics, 243 (1-2), 93–105.
[42] El-Shabouri, M. H. (2002). Positively charged nanoparticles for improving the oral
bioavailability of cyclosporin-A. International journal of pharmaceutics, 249 (1-2), 101-108.
[43] Sonaje, K.; Lin, K. J.; Tseng, M. T.; Wey, S. P.; Su, F. Y.; Chuang, E. Y.; Hsu, C. W.; Chen,
C. T. & Sung, H. W. (2011). Effects of chitosan-nanoparticle-mediated tight junction opening
on the oral absorption of endotoxins. Biomaterials, 32 (33), 8712–8721.
[44] Cui, Z. & Mumper, R. J. (2001). Chitosan-based nanoparticles for topical genetic
immunization. Journal of Controlled Release, 75 (3), 409–419.
[45] Cui, W.; Lu, X.; Cui, K.; Wu, J.; Wei, Y. & Lu, Q. (2011). Fluorescent nanoparticles of
chitosan complex for real-time monitoring drug release. Langmuir, 27 (13), 8384–8390.
[46] Shen, J. M.; Xu, L.; Lu, Y.; Cao, H. M.; Xu, Z. G.; Chen, T. & Zhang, H. X. (2012). Chitosan-
based luminescent/magnetic hybrid nanogels for insulin delivery, cell imaging, and
antidiabetic research of dietary supplements. International journal of pharmaceutics, 427 (2),
400–409.
[47] Moon, H. J.; Lee, J. S.; Talactac, M. R.; Chowdhury, M. Y.; Kim, J. H.; Park, M. E.; Choi, Y.
K.; Sung, M. H. & Kim, C. J. (2012). Mucosal immunization with recombinant influenza
hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection
against highly pathogenic influenza A virus. Veterinary microbiology, 160 (3-4), 277-289.
[48] Shi, Z. L.; Neoh, K. G.; Kang, E. T. & Wang, W. (2006). Antibacterial and mechanical
properties of bone cement impregnated with chitosan nanoparticles. Biomaterials, 27, 2440–
2449.
[49] Chen, H.-J.; Zhanga, Z.-H.; Luo, L.-J. & Yao, S.-Z. (2012). Surface-imprinted chitosan-coated
magnetic nanoparticles modifiedmulti-walled carbon nanotubes biosensor for detection of
bovine serum albumin. Sensors and Actuators B, 163, 76–83.
[50] Monier, M. (2012). Adsorption of Hg2+, Cu2+ and Zn2+ ions from aqueous solution using
formaldehyde cross-linked modified chitosan-thioglyceraldehyde Schiff's base. International
Journal of Biological Macromolecules, 50 (3), 773– 781.
[51] Fan, L.; Zhang, Y.; Luo, C.; Lu, F.; Qiu, H. & Sun, M. (2012). Synthesis and characterization
of magnetic β-cyclodextrin-chitosan nanoparticles as nano-adsorbents for removal of methyl
blue. International journal of biological macromolecules, 50 (2):444–450.
[52] Llanes, F.; Ryan, D. H. & Marchessault, R. H. (2000). Magnetic nanostructured composites
using alginates of different M/G ratios as polymeric matrix. International Journal of Biological
Macromolecules, 27 (1), 35–40.
[53] Wang, Q.; Zhang, D.; Xu, H.; Yang, X.; Shen, A. Q.; Yang, Y. (2012). Microfluidic one-step
fabrication of radiopaque alginate microgels with in situ synthesized barium sulfate
nanoparticles. Lab on a chip, 12 (22), 4781–4786.
76
[54] González-Rodríguez, M. L.; Holgado, M. A.; Sánchez-Lafuente, C.; Rabasco, A. M. & Fini,
A. (2002). Alginate/chitosan particulate systems for sodium diclofenac release. International
Journal of Pharmaceutics, 232 (1-2), 225–234.
[55] De, S. & Robinson, D. (2003). Polymer relationships during preparation of chitosan-alginate
and poly-l-lysine-alginate nanospheres. Journal of Controlled Release, 89 (1), 101–112.
[56] Cheng, Y.; Yu, S.; Wang, J.; Qian, H.; Wu, W. & Jiang, X. (2012). In vitro and in vivo
Antitumor activity of doxorubicin-loaded alginic-acid-based nanoparticles. Macromolecular
bioscience, 12 (10), 1326–1335.
[57] Nagarwal, R. C.; Kumar, R. & Pandit, J. K. (2012). Chitosan coated sodium alginate–chitosan
nanoparticles loaded with 5-FUfor ocular delivery: In vitro characterization and in vivo study
in rabbit eye. European Journal of Pharmaceutical Sciences, 47, 678–685.
[58] Lin, S.; Huang, R.; Cheng, Y.; Liu, J.; Lau, B. L. T. & Wiesner, M. R. (2012). Silver
nanoparticle-alginate composite beads for point-of-use drinking water disinfection. Water
Research, 1–7.
77
5.3. ARTIGO 2
78
79
80
81
82
83
5.4. ARTIGO 3
84
85
86
87
88
89
90
91
Almeida-Lima J. PPGCSA/CCS
6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES
O grupo em que está inserido este trabalho tem estudado há algumas
décadas as atividades biológicas/farmacológicas presentes em fucanas em
diversas espécies de algas, como os polissacarídeos de alga. Este projeto de
pesquisa inicialmente teve como objetivo inovar nas pesquisas com
polissacarídeos e desenvolver uma nova linha de pesquisa na área da
nanomedicina propondo-se a sintetizar nanogéis de fucana, além da inserção
de novas técnicas as metodologias já utilizadas no laboratório (BIOPOL).
Os estudos mais avançados se concentraram em torno da fucana A
obtidas da alga S. schröederi. Esse polímero em pesquisas anteriores tem
apresentado uma excelente atividade biológica, especialmente pela alta
atividade antitumoral. Além disso, o material biológico a ser utilizado é de fácil
obtenção nas quantidades desejadas para o desenvolvimento deste trabalho, o
que não foi empecilho para o pleno trabalho das atividades propostas no início
da pesquisa. O laboratório também fez colaboração com o Prof. Miguel Gama,
da Universidade do Minho (Portugal), que tem experiência com nanopartículas
sintetizadas a partir de polissacarídeos, e que ajudou o nosso grupo na
caracterização dos nanogéis obtidos. Além disso, tivemos também a
colaboração da Profa. Helena Nader da UNIFESP, que se comprometeu a nos
auxiliar nos experimentos ou na utilização de equipamentos de não
disponibilidade aqui. Outros departamentos da UFRN das mais diversas áreas
(Física, Química, Engenharia de Materiais, Farmácia, Genética e Instituto do
Cérebro) também foram utilizados para a utilização de equipamentos como
também para o desenvolvimento de técnicas novas.
A produção de nanogéis de fucana foi desafiadora, já que fomos o
primeiro grupo a iniciar as pesquisas com nanogéis no Departamento de
Bioquímica da UFRN, mas foi compensadora, pois com o desenvolvimento
desse projeto pode-se iniciar uma fase nova na pesquisa na tentativa de
elucidar cada vez mais os mecanismos de ação pelos quais ela age, e
consequentemente, a longo prazo começar a fazer o registro de patentes.
Ao longo do desenvolvimento desta pesquisa, o cronograma de
atividades seguiu dentro dos padrões esperados e todas as dificuldades
92
Almeida-Lima J. PPGCSA/CCS
encontradas foram superadas, através da ajuda dos colaboradores de diversos
centros de pesquisa, e inclusive do próprio laboratório onde se realizou a
pesquisa (BIOPOL/UFRN), com o auxílio de um aluno da iniciação científica
(Arthur Vidal), quando tive oportunidade de orientá-lo durante parte dos
experimentos.
Também considero importante ressaltar que durante processo de
formação participei de alguns Congressos, Simpósios e Encontros científicos
internacionais, nacionais e regionais, como autora ou em coautoria de
aproximadamente 20 resumos científicos. Destaco a minha participação no 4º.
International Symposium in Biochemistry of Macromolecules and Biotechnology
em 2012, quando um dos trabalhos em que sou coautora recebeu a Menção
Honrosa Prêmio Marcionilo Lins. Também destaco alguns eventos mais
relevantes: XLII Annual Meeting of the Brazilian Society for Biochemistry and
Molecular Biology – SBBq (2013) e 62ª Reunião Anual da SBPC (2010).
Até o momento tenho 13 artigos publicados em revista científicas, sendo
dois como primeira autora e os demais dividindo coautoria. As revistas foram:
Holos (IFRN), Publica (UFRGN) Revista Brasileira de Farmacognosia,
Toxicology Letters, Journal of Applied Toxicology, Biomedicine &
Pharmacotherapy, Carbohydrate Polymers, Marine Drugs, Molecules e Journal
of Applied Phycology. Além disso, também fiz parte na elaboração do capítulo
de livro na Marine Medicinal Glycomic e de uma banca examinadora de
trabalho de conclusão de curso (monografia).
O desenvolvimento deste estudo me proporcionou a oportunidade de
interagir com diversos ramos da pesquisa científica, bem como a aprendizagem
de algumas técnicas, o que conferiu um caráter interdisciplinar ao trabalho.
Como projeto para o futuro, planejamos continuar com os estudos na mesma
linha de pesquisa, em um futuro pós-doutorado.
93
Almeida-Lima J. PPGCSA/CCS
7. REFERÊNCIAS 1. WHO. Global Status Report on Non-Communicable Diseases 2010.
Disponível em:
http://www.who.int/nmh/publications/ncd_report2010/en/.>Accesso em: Feb
05, 2014.
2. Bray F, Jemal A, Grey N, Ferlay J, Forman D. Global cancer transitions
according to the Human Development Index (2008–2030): a population-
based study. The Lancet Oncology 2012; 13(8):790–801.Rang et al., 2004
3. Rang HP, Dale MM, Ritter JM, Moore PK. Farmacologia. 5ed. Rio de
Janeiro: Elsevier 2004, p. 789-809.
4. Rothenberg ML, Carbone DP, Johnson DH. Improving the evaluation of new
cancer treatments: challenges and opportunities. Nat Rev Cancer 2003;
3(4):303-9.
5. Melo-Silveira RF, Almeida-Lima J, Rocha HAO. Application of marine
polysaccharides in natechnology. In: Vitor Hugo Pomin. (Org.). Marine
Medicinal Glycomics. 1ed. New York: Nova Science, 2013, v. 01, p. 65-114.
6. Freitas Jr R A. Nanotechnology, nanomedicine and nanosurgery.
International Journal of Surgery 2005; 3(4):243-246.
7. Kabanov AV, Vinogradov SV. Nanogels as pharmaceutical carriers: finite
networks of infinite capabilities. Angew Chem Int Ed Engl 2009;
48(30):5418-29
8. Gonçalves C, Pereira P, Gama M. Self-Assembled Hydrogel Nanoparticles
for Drug Delivery Applications. Materials 2010; 3:1420-60
9. Zong A, Cao H, Wang F. Anticancer polysaccharides from natural
resources: a review of recent research. Carbohydr Polym 2012; 90(4):1395-
410.
10. Rocha HAO, Franco CRC, Trindade ES, Veiga SS, Leite EL, Nader HB,
Dietrich CP. Fucan inhibits Chinese hamster ovary cell (CHO) adhesion to
fibronectin by binding to the extracellular matrix. Planta Medica (Stuttgart)
2005; 71(7):628-33.
11. Rocha HAO, Leite EL, Medeiros VP, Lopes CC, Nascimento FD, Tersariol
ILS, Sampaio LO, Nader HB. Natural sulfated polysaccharides as
antithrombotic compounds. Structural characteristics and effects on the
coagulation cascade. In Carbohydrate Structure and Biological Function.
Kerala: Transworld Research Network, 2006.
94
Almeida-Lima J. PPGCSA/CCS
12. Leite EL, Medeiros MGL, Rocha HAO, Farias GGM, Silva LF, Chavante SF,
et al. Structure of a new fucan from the algae Spatoglossum schröederi.
Plant Sci 1998; 132: 215-28.
13. Barroso EM, Costa LS, Medeiros VP, Cordeiro SL, Costa MSP, Franco CR,
et al. A non-anticoagulant heterofucan has antithrombotic activity in vivo.
Planta Med 2008; 74:712–8.
14. Almeida-Lima J, Dantas-Santos N, Gomes DL, Cordeiro SL, Sabry DA,
Costa LS, et al. Evaluation of acute and subchronic toxicity of a non-
anticoagulant, but antithrombotic algal heterofucan from the Spatoglossum
schröederi in Wistar rats. Rev Bras Farmacogn 2011; 21:674–9
15. Almeida-Lima J, Costa LS, Silva NB, Melo-Silveira RF, Silva FV, Felipe MB,
et al. Evaluating the possible genotoxic, mutagenic and tumor cell
proliferation-inhibition effects of a non-anticoagulant, but antithrombotic algal
heterofucan. J Appl Toxicol. 2010; 30:708-15.
16. Berry D, Lynn DM, Sasisekharan R, Langer R. Poly (beta-amino ester)s
promote cellular uptake of heparin and cancer cell death. Chem Biol 2004;
11(4):487-98.
17. Yu MK, Lee DY, Kim YS, Park K, Park SA, Son DH et al. Antiangiogenic and
apoptotic properties of a novel amphiphilic folate-heparin-lithocholate
derivative having cellular internality for cancer therapy.Pharm Res 2007;
24:705-14.
18. Bae KH, Mok H, Park TG. Synthesis, characterization, and intracellular
delivery of reducible heparin nanogels for apoptotic cell death. Biomaterials
2008; 29(23):3376-83.
19. Dantas-Santos N, Almeida-Lima J, Vidal AA, Gomes DL, Oliveira RM,
Santos Pedrosa S, et al. Antiproliferative activity of fucan nanogel. Mar
Drugs 2012;10:2002-22.
20. Dietrich CP, Farias GGM, Abreu LRD, Leite EL, Silva LF, Nader HBA. A new
aproach for characterization of polysaccharides from algae: presence of four
main acidic pollysaccharides in three specie of the class Phaeophycea.
Plant Sci 1995; 108:143-53.
21. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric
method for determination of sugars, and related substances. Anal Chem
1956; 28:350–356.
22. Mosmann T. Rapid colorimetric assay for cellular growth and survival. J
Immunol Methods 1983; 65:55-63.
95
Almeida-Lima J. PPGCSA/CCS
23. Costa LS, Fidelis GP, Cordeiro SL, Oliveira RM, Sabry DA, Câmara RB,
Nobre LT, Costa MS, Almeida-Lima J, Farias EH, Leite EL, Rocha HA.
Biological activities of sulfated polysaccharides from tropical seaweeds.
Biomed Pharmacother 2010; 64(1):21-8
24. Vinayak RC, Sabu AS, Chatterji A. Bio-prospecting of a few brown
seaweeds for their cytotoxic and antioxidant activities. Evid Based
Complement Alternat Med. 2011; 2011:673083.
25. Dreyfuss JL, Regatieri CV, Lima MA, Paredes-Gamero EJ, Brito AS,
Chavante SF, Belfort R Jr, Farah ME, Nader HB. A heparin mimetic isolated
from a marine shrimp suppresses neovascularization. J Thromb Haemost.
2010; 8(8):1828-37.
96
Almeida-Lima J. PPGCSA/CCS
ANEXOS
97
Almeida-Lima J. PPGCSA/CCS
8.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)
98
Almeida-Lima J. PPGCSA/CCS
99
Almeida-Lima J. PPGCSA/CCS
100
Almeida-Lima J. PPGCSA/CCS
8.2. NORMAS DA REVISTA PARA SUBMISSÃO (MARINE DRUGS)
Marine Drugs — Instructions for Authors
Please first read the section 'Aims & Scope' to have an overview, and to assess if your
manuscript is suitable for this journal.
Shortcuts
Submission
Manuscript Preparation
Authorship and Authors Contributions
Correct Identification of Components of Natural Products
Ethics Approval of Research
Potential Conflicts of Interest
Peer-Review and Editorial Procedure
Review / Referees
English Corrections
MDPI Publication Ethics Statement
Supplementary Material
Please use the MS Word template or LaTeX template to prepare your paper.
Submission of Manuscripts
Submission Process: Manuscripts for Marine Drugs should be submitted online
at susy.mdpi.com. To submit your manuscript, register and log in to this website. Once
you are registered, click here to go to the submission form for Marine Drugs.
Accepted File Formats:
o Microsoft Word: Manuscript prepared in MS Word must be converted into a
single file before submission. When preparing manuscripts in MS Word,
the Marine Drugs Microsoft Word template file must be used. Please do not
insert any graphics (schemes, figures, etc.) into a movable frame which can
superimpose the text and make the layout very difficult.
o LaTeX: Manuscripts prepared in LaTeX must be zipped into one ZIP folder
(include all source files and images, so that the Editorial Office can modify the
manuscript before peer-review, if needed). Ensure to send a copy of your
manuscript as a PDF file also, if you decided to use LaTeX. When preparing
manuscripts in LaTeX, please use the Marine DrugsLaTeX template files.
Coverletter: Please provide a short cover letter where you detail the reasons why the
editors of Marine Drugs should consider your paper for publication in this journal. Check
in your cover letter whether you supplied at least 5 possible referees. Check if
the English corrections are done before submission.
Note Regarding Conference Papers: Expanded and high quality conference papers
are also considered in Marine Drugs if they fulfill the following requirements: (1) The
paper should be expanded to the size of a research article. (2) The conference paper
should be cited and mentioned as a note on the first page of the paper. (3) If the
authors do not hold the copyright to the published conference paper, authors should
seek the appropriate permission from the copyright holder. (4) Authors are asked to
101
Almeida-Lima J. PPGCSA/CCS
disclose the conference paper in their cover letter including a statement on what has
been changed compared to the conference paper.
Manuscript Preparation
Paper Format: A4 paper format, the printing area is 17.5 cm x 26.2 cm. The margins
should be 1.75 cm on each side of the paper (top, bottom, left, and right sides). There is
no page limit. Full experimental details (for original research papers) must be provided
so that the results can be reproduced.
Formatting / Style: The paper style of Marine Drugs should be followed. You may
download a template file from the Marine Drugs homepage to prepare your paper. It is
not necessary to follow the manuscript structure showed in the template file for review
papers.
Authors List and Affiliation Format: Authors' full first and last names must be given.
Abbreviated middle name can be added. For papers written by various contributors a
corresponding author must be designated. The PubMed/MEDLINE format is used for
affiliations: complete address information including city, zip code, state/province,
country, and email address should be added. All authors who contributed significantly to
the manuscript (including writing a section) should be listed on the first page of the
manuscript, below the title of the article. Other parties, who provided only minor
contributions, should be listed under Acknowledgments only. A minor contribution might
be a discussion with the author, reading through the draft of the manuscript, or
performing English corrections.
Abstract and Keywords: The abstract should be prepared as one paragraph of about
200 words. For research articles, abstracts should give a pertinent overview of the work,
its purpose, the main methods or treatments applied; summarize the article's findings or
facts and indicate the authors' conclusions or interpretation. As such, the abstract aims
at being an objective representation of the article and must not contain results or data
which are not presented and substantiated in the main text. Note that abstracts serve
two main purposes: on one hand abstracts are used by potential readers to assess the
relevancy of an article for their own work. On the other hand, abstracts are used by
indexing databases to catalog articles appropriately. Also, three to 10 pertinent
keywords need to be added after the abstract. We recommend that the abstract and the
keyword list use words that are specific to the article yet reasonably common within the
subject discipline.
Abstract Graphic: Authors are encouraged to provide a self-explanatory graphical
abstract of the paper to be used along with the abstract on the Table of Contents and
search results. The graphic should not exceed 550 pixels width/height and can be
provided as a PDF, JPG, PNG or GIF file. The minimum text size in the graphic should
be 12 pt.
Units: SI units (International System of Units) should be used for this journal. Imperial,
US customary and other units should be converted to SI units whenever possible before
submission of a manuscript to the journal.
Figures, Schemes and Tables: Authors are encouraged to prepare figures and
schemes in color. Full color graphics will be published free of charge. We kindly request
authors to provide figures and schemes at a sufficiently high resolution (min. 600 pixels
width, 300 ppi). Figures and schemes must be numbered (Figure 1, Scheme I, Figure 2,
Scheme II, etc.) and a explanatory title must be added. Tables should be inserted into
the main text, and numbers and titles for all tables supplied. All table columns should
have an explanatory heading. To facilitate the copy-editing of larger tables, smaller
fonts may be used, but in no case should these be less than 10 pt in size. Authors
should use the Table option of MS Word to create tables, rather than tabs, as tab
102
Almeida-Lima J. PPGCSA/CCS
delimited columns are often difficult to format for the final PDF output. Please supply
captions for all figures, schemes and tables. The captions should be prepared as a
separate paragraph of the main text and placed in the main text before a table, a figure
or a scheme.
Equations: If you are using Word, please use either the Microsoft Equation Editor or
the MathType add-on in your paper. It should be editable, not in the format of a picture.
Chemical Structures and Reaction Schemes: Chemical structures and reaction
schemes should be drawn using an appropriate software package designed for this
purpose. As a guideline, these should be drawn to a scale such that all the details and
text are clearly legible when placed in the manuscript (i.e. text should be no smaller that
8-9 pt.). To facilitate editing we recommend the use of any of the software packages
widely available for this purpose: MDL® Isis/Draw, ACD/ChemSketch®, CS
ChemDraw®, ChemWindow®, etc. Free versions of some of these products are
available for personal or academic use from the respective publishers. If another less
common structure drawing software is used, authors should ensure the figures are
saved in a file format compatible with of one of these products.
Physical and Spectroscopic Data: Physical and spectroscopic data as well as tables
for NMR data should be prepared according to the ACS's Preparation and Submission
of Manuscripts standard (page 4).
Experimental Data: To allow for correct abstracting of the manuscripts all compounds
should be mentioned by correct chemical name, followed by any numerals used to refer
to them in the paper. The use of the IUPAC nomenclature conventions is preferred,
although alternate naming systems (for example CAS rules) may be used provided that
a single consistent naming system is used throughout a manuscript. For authors
perhaps unfamiliar with chemical nomenclature in English we recommend the use of
compound naming software such as AutoNom. Full experimental details must be
provided, or, in the case of many compounds prepared by a similar method, a
representative typical procedure should be given. The general style used in the Journal
of Organic Chemistry is preferred. Complete characterization data must be given for all
new compounds. For papers mentioning large numbers of compounds a tabular format
is acceptable. For known compounds appropriate literature references must be given.
Conflicts of Interest: Authors must identify and declare any personal circumstances or
interest that may be perceived as inappropriately influencing the representation or
interpretation of reported research results. If there is no conflict of interest, please state
"The authors declare no conflict of interest." This should be conveyed in a separate
"Conflicts of Interest" statement immediately preceding the "References" section of the
manuscript. Financial support for the study must be fully disclosed under the
"Acknowledgments" section.
Acknowledgments: Please clearly indicate grants that you have received in support of
your research work (including funds for covering the costs to publish in open access).
Note that some funders will not refund article processing charges (APC) if the funder
and grant number are not clearly identified in the paper. The Acknowledgments section
is placed just before the References section.
References: Please ensure that a comprehensive list of all relevant references is
provided at the end of the manuscript, and that all references are cited within the paper
and numbered consecutively throughout the paper (including citations in tables and
legends). References should preferably be prepared with a bibliography software
package, such as Zotero, EndNote orReferenceManager. If references are prepared
manually they must be checked for integrity and correctness.
Reference Formatting: All the references mentioned in the text should be listed
separately and as the last section at the end of the manuscript, and be numbered
consecutively throughout the paper. Do not repeat references in the references list.
103
Almeida-Lima J. PPGCSA/CCS
Reference numbers should be placed in square brackets [ ], and placed before the
punctuation; for example [4] or [1-3]. For embedded citations in the text with pagination,
use both parentheses and brackets to indicate the reference number and page
numbers; for example [5] (p. 10). or [6] (pp. 101–105). Include the full title for cited
articles. See the Reference Preparation Guide for more detailed information.
Supplementary Material and Research Data: authors are encouraged to make their
experimental and research data openly available. Large datasets and files should be
deposited to specialized data repositories. Small datasets, spreadsheets, images, video
sequences, conference slides, software source code, etc. can be included with the
submission and published as supplementary material. Please read the information
about Supplementary Material and Data Deposit beneath for additional information and
instructions.
Authorship and Authors Contributions
For research articles with more than one author, authors are asked to prepare a short, one
paragraph statement giving the individual contribution of each co-author to the reported
research and writing of the paper. The paragraph should be titled "Author Contributions" and
placed in the paper after the "Acknowledgement" section and before the "Conflicts of Interest"
statement.
Only major contributors should be listed as authors. Those with small or technical contributions
can be mentioned in the Acknowledgements section. Authors themselves are responsible for
the correct identification and attribution of authorship. According to the COPE standard, to
which this journal adheres, "all authors should agree to be listed and should approve the
submitted and accepted versions of the publication. Any change to the author list should be
approved by all authors including any who have been removed from the list. The corresponding
author should act as a point of contact between the editor and the other authors and should
keep co-authors informed and involve them in major decisions about the publication (e.g.
responding to reviewers’ comments)." [1]
1. Wager, E.; Kleinert, S. Responsible research publication: international standards for
authors. A position statement developed at the 2nd World Conference on Research
Integrity, Singapore, July 22-24, 2010. In Promoting Research Integrity in a Global
Environment; Mayer, T., Steneck, N., eds.; Imperial College Press / World Scientific
Publishing: Singapore; Chapter 50, pp. 309-16.
Correct Identification of Components of Natural Products
The correct identification of the various components of extracts from natural sources is of key
importance, and as publishers we are keenly aware of our responsibility to the scientific
community in this area. Consequently, for papers on this topic, we have adopted the
recommendations of the Working Group on Methods of Analysis of the International
Organization of the Flavour Industry (IOFI), as published in Flavour Fragr. J. 2006, 21, 185.
These recommendations may be summarized as follows:
Any identification of a natural compound must pass scrutiny by the latest forms of available
analytical techniques. This implies that its identity must be confirmed by at least two different
methods, for example, comparison of chromatographic and spectroscopic data (including mass,
IR and NMR spectra) with those of an authentic sample, either isolated or synthesized. For
papers claiming the first discovery of a given compound from a natural source, the authors must
104
Almeida-Lima J. PPGCSA/CCS
provide full data obtained by their own measurements of both the unknown and an authentic
sample, whose source must be fully documented. Authors should also consider very carefully
potential sources of artifacts and contaminants resulting from any extraction procedure or
sample handling.
Ethics Approval of Research
Manuscripts containing original descriptions of research conducted in humans or experimental
animals must contain details of approval by a properly constituted research ethics committee.
As a minimum, the project identification code, date of approval and name of the ethics
committee or institutional review board should be cited in the Experimental section.
Potential Conflicts of Interest
It is the authors' responsibility to identify and declare any personal circumstances or interests
that may be perceived as inappropriately influencing the representation or interpretation of
clinical research. If there is no conflict, please state "The authors declare no conflict of interest.".
This should be conveyed in a separate "Conflicts of Interest" statement preceding the
"Acknowledgments" and "References" sections at the end of the manuscript. Financial support
for the study must be fully disclosed under the "Acknowledgments" section.
105
Almeida-Lima J. PPGCSA/CCS
8.3. DECLARAÇÃO
DECLARAÇÃO DE CORREÇÃO DE PORTUGUÊS
106
Almeida-Lima J. PPGCSA/CCS
8.4. COMITÊ DE ÉTICA